Discovery of a Small-Molecule Degrader of Bromodomain and Extra-Terminal (BET) Proteins with Picomolar Cellular Potencies and Capable of Achieving Tumor Regression

Article

pubs.acs.org/jmc

Discovery of a Small-Molecule Degrader of Bromodomain and Extra-

Terminal (BET) Proteins with Picomolar Cellular Potencies and

Capable of Achieving Tumor Regression

Bing Zhou,^†,‡,∇Jiantao Hu,^†,‡,∇Fuming Xu,^†,‡,∇Zhuo Chen,^†,‡,∇Longchuan Bai,^†,‡,∇

Ester Fernandez-Salas,^†,§,∇Mei Lin,^†,‡,∇Liu Liu,^†,‡Chao-Yie Yang,^†,‡Yujun Zhao,^†,‡

Donna McEachern,^†,‡Sally Przybranowski,^†,‡Bo Wen,^†,∥Duxin Sun,^†,∥and Shaomeng Wang*^{,†,‡,⊥,#}

‡

§

∥

^†University of Michigan Comprehensive Cancer Center, and Departments of Internal Medicine, Pathology, Pharmaceutical

⊥

#

Sciences, Medicinal Chemistry, and Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, United States

S

* Supporting Information

ABSTRACT: The bromodomain and extra-terminal (BET) family

proteins, consisting of BRD2, BRD3, BRD4, and testis-speciﬁc

BRDT members, are epigenetic “readers” and play a key role in the

regulation of gene transcription. BET proteins are considered to be

attractive therapeutic targets for cancer and other human diseases.

Recently, heterobifunctional small-molecule BET degraders have

been designed based upon the proteolysis targeting chimera

(PROTAC) concept to induce BET protein degradation. Herein,

we present our design, synthesis, and evaluation of a new class of

PROTAC BET degraders. One of the most promising compounds,

23, eﬀectively degrades BRD4 protein at concentrations as low as 30

pM in the RS4;11 leukemia cell line, achieves an IC₅₀value of 51 pM in inhibition of RS4;11 cell growth and induces rapid tumor

regression in vivo against RS4;11 xenograft tumors. These data establish that compound 23 (BETd-260/ZBC260) is a highly

potent and eﬃcacious BET degrader.

transcription. In addition to small-molecule BET inhibitors, a

new approach has recently been developed to target BET

proteins for degradation based upon the proteolysis targeting

chimera (PROTAC) concept.²⁰In this approach, a hetero-

bifunctional (chimeric) molecule is designed to contain a BET

inhibitor, which binds to BET proteins, another small-molecule

ligand, which binds to an E3 ubiquitin ligase complex, and a

linker to tether these two ligands together.^21,22A number of

INTRODUCTION

■

Bromodomain-containing proteins are epigenetic “readers”. By

binding to acetylated lysine residues on the histone tails,

bromodomain-containing proteins play a key role in regulation

of gene transcription.¹Among bromodomain-containing

proteins, the bromodomain and extra-terminal domain (BET)

family of proteins, consisting of BRD2, BRD3, BRD4, and

testis-speciﬁc BRDT members, have emerged as exciting new

therapeutic targets for cancer and other human diseases.²⁻⁶

The discovery of 1 ((+)-JQ-1) as the ﬁrst potent and selective

BET inhibitor (Figure 1) has greatly promoted investigations of

BET proteins as new therapeutic targets in human cancers and

other diseases.⁷Several BET inhibitors such as 2 (OTX015)

and 3 (I-BET762) (Figure 1) have been advanced into clinical

development.⁸⁻¹³Recently, preliminary clinical data have

provided an important clinical proof-of-concept that BET

inhibition has therapeutic potential for the treatment of certain

forms of human cancer, including NUT (nuclear protein in

testis) midline carcinoma, multiple myeloma, and acute

myeloid leukemia (AML).^7,10,11,14Preclinical studies have also

suggested that BET inhibitors may have therapeutic potential

for the treatment of other human cancers, as well as other

human diseases and conditions.¹⁵⁻¹⁹

BET degraders have been reported, including 4 (dBET1),²³

5

(ARV-771),²⁴6 (ARV-825),²⁵and MZ1²⁶(Figure 1).

Compounds 4 and 6 were designed using 1 or 2, two closely

related BET inhibitors, and thalidomide, which is a ligand for

cereblon, a component of the Cullin4A ubiquitin ligase

complex.^23,25In comparison, BET degrader 5 was designed

using 1 for the BET inhibitor portion and a ligand for the von

Hippel−Lindau E3 ubiquitin ligase.²⁴These BET degraders

have been shown to eﬃciently induce BET protein degradation

and to be more potent in inhibition of cancer cell growth and in

induction of apoptosis than their corresponding BET inhibitors.

Compound 4 is more eﬀective in inhibition of tumor growth in

Special Issue: Inducing Protein Degradation as a Therapeutic

Strategy

Small-molecule BET inhibitors are designed to bind the BET

bromodomains and to block the interaction of BET proteins

with acetylated lysine residues on histone tails to regulate gene

Received: December 11, 2016

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Figure 1. Chemical structures of representative BET inhibitors 1, 2, 3, 7, 8 and three representative previously reported BET degraders 4, 5, and 6.

Table 1. Binding Aﬃnities of BET Inhibitors

a

IC₅₀values were obtained from three separate experiments.

vivo than 1 in an acute leukemia model in mice,²³and

compound 5 achieves eﬀective inhibition of tumor growth in a

castration-resistance prostate cancer xenograft model in mice.²⁴

Collectively, these studies provide evidence that small-molecule

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Figure 2. (A) Cocrystal structure of BRD4 BD2 complexed with 7 (green, PDB code 4Z93). (B) Modeled structure of BRD4 BD2 complexed with

8 (yellow). Water molecules are shown as red spheres. Hydrogen bonds are depicted in dashed lines. Residues in BRD4 BD2 close to the proposed

linkage site of 8 are labeled in red.

Table 2. Optimization of Linker Length and Composition

a

IC₅₀values were obtained from three independent experiments.

degraders of BET proteins may have a promising therapeutic

potential for the treatment of human cancers and potentially

other diseases and conditions.

Recently, our laboratory reported the discovery of

azacarbazoles as a new class of potent and selective BET

bromodomain inhibitors.²⁷In the present study, we report the

discovery of a new class of small-molecule BET degraders

designed based upon our azacarbazole-based BET inhibitors

and thalidomide/lenalidomide as ligands for cereblon/Cull-

in4A. Through extensive optimization of the linker region, we

have obtained a series of highly potent BET degraders. Among

these, compound 23 (BETd-260) is capable of inducing

degradation of BRD2, BRD3, and BRD4 proteins at 30−100

pM in the RS4;11 leukemia cells. Compound 23 achieves an

IC₅₀value of 51 pM in inhibition of the RS4;11 cell growth and

induces rapid tumor regression of the RS4;11 xenograft tumors

with no signs of toxicity in mice. Compound 23 is a highly

potent and eﬃcacious BET degrader and warrants extensive

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evaluation for the treatment of human cancers and other

diseases.

than 10. These data demonstrate that both the length and the

composition of the linker have a considerable inﬂuence on the

cellular potencies of the resulting BET degraders.

RESULTS AND DISCUSSION

We next sought to determine the optimal linker length in 12

for cellular potencies by shortening the linker by one methylene

group progressively, which resulted in compounds 13−17

(Table 2). Compound 13 with a linker one methylene group

shorter than that in 12 is 3 times less potent than 12. However,

compound 14 with two methylene groups shorter than 12 in

the linker achieves an IC₅₀value of 0.14 nM in inhibition of

RS4;11 cell growth and is slightly more potent than 12.

Shortening the linker in 14 by one additional methylene group

resulted in 15, which is ∼3 times less potent than 14 in

inhibition of RS4;11 cell growth, and further shortening the

linker in 15 by one methylene or ethylene group yielded 16 or

17, respectively, which is 5 and 10 times less potent than 15,

respectively, in inhibition of RS4;11 cell growth. These data

demonstrate that for achieving the most potent cell growth

inhibition activity, an optimal linker length should comprise

-(CH₂)₄₋₇NH- in these BET degraders, as is shown in

compounds 12−15. When the linker becomes too short as

in, for examples, compounds 16 and 17, the cellular potency is

greatly decreased.

To investigate whether the SAR results obtained in the

RS4;11 cell line are valid in a diﬀerent cell line, we evaluated

this series of compounds together with 4 and 6 as control

compounds for their cell growth inhibitory activity in the

MOLM-13 leukemia cell line, which harbors a mixed lineage

leukemia protein 1 (MLL1) fusion gene and is also responsive

to BET inhibitors in our previous study.²⁷The data obtained

are summarized in Table 2. In general, the IC₅₀values obtained

for all these BET degraders in the MOLM-13 cell line are 5−10

times higher than those obtained in the RS4;11 cell line.

Compounds 6 and 4 have IC₅₀values of 18.2 nM and 657 nM,

respectively, in inhibition of MOLM-13 cell growth and are 5.5

and 8.3 times less potent than those in the RS4;11 cell line. We

obtained a very similar SAR result for BET degraders 9−17 in

the MOLM-13 cell line when compared to that in the RS4;11

cell line. Compounds 12 and 14 are two of the most potent

BET degraders in this series with IC₅₀values of 1.2 nM and 2.1

nM, respectively, in inhibition of MOLM-13 cell growth.

Having determined the optimal linker length, we next

performed further modiﬁcations of the linker composition in

compound 14 (Table 3). Replacement of the NH group in 14

with a methylene group yielded 18, which with an IC₅₀value of

0.17 nM in inhibition of RS4;11 cell growth is as potent as 14.

Replacement of one methylene group in 18 with an oxygen

atom generated 19, which has an IC₅₀value of 0.42 nM in

inhibition of RS4;11 cell growth and is 2 times less potent than

18. Shortening the linker in 18 by one methylene group

resulted in 20 which, with an IC₅₀value of 0.14 nM in

inhibition of RS4;11 cell growth, is equipotent to 14. Thus,

modiﬁcations of the linker compositions identiﬁed compounds

18 and 20 as two very potent BET degraders with

subnanomolar IC₅₀values in inhibition of RS4;11 cell growth.

In all of the above synthesized compounds, we employed

thalidomide as the ligand for cereblon. Lenalidomide has been

developed as a second generation thalidomide analogue for the

treatment of multiple myeloma and myelodysplastic syn-

dromes.²⁸Although thalidomide and lenalidomide have similar

binding aﬃnities to cereblon,²⁹they may have diﬀerent cell

permeability and/or pharmacokinetic proﬁles, and thus we

investigated the eﬀect of replacing thalidomide with lenalido-

■

Starting from our previously reported BET inhibitor 7²⁷

(Figure 1), we performed further optimization for this class of

BET inhibitors and identiﬁed 8 (HJB97) as a high-aﬃnity BET

inhibitor. In our FP-based competitive binding assays, 8 binds

to BRD2, BRD3, and BRD4 with high aﬃnities (K_i< 1 nM)

and is >10 times more potent than 1 or 2 (Table 1).

Compound 8 also potently inhibits cell growth in RS4;11 and

MOLM-13 acute leukemia cell lines, which are known to be

sensitive to BET inhibitors. Hence, compound 8 is a potent

BET inhibitor and was employed in our design of BET

degraders.

Upon the basis of the cocrystal structure of BRD4 BD2

complexed with 7 (PDB code 4Z93), we modeled the structure

of BRD4 BD2 complexed with 8 (Figure 2). Our modeled

structure showed that the 2-carboxamide group attached to the

[6,5,6] tricyclic system in 8 is exposed to solvent, making it a

suitable site for tethering to thalidomide/lenalidomide for the

design of potential PROTAC degraders of BET proteins

(Figure 2). Accordingly, we designed and synthesized 9 as a

potential BET degrader using 8 for the BET inhibitor portion,

thalidomide as the cereblon ligand, and the same linker that was

used in 4. In a cell growth assay, the BET degrader 9 has an

IC₅₀value of 4.3 nM in the RS4;11 acute leukemia cell line and

is 6 times more potent than the corresponding BET inhibitor 8

(Table 2). Western blotting analysis showed that 9, at

concentrations as low as 3−10 nM, is eﬀective in decreasing

the level of BRD2, BRD3, and BRD4 proteins in the RS4;11

cells whereas the BET inhibitor 8 at both 100 and 300 nM fails

to decrease the level of BRD2−4 proteins (Figure 3). Two

Figure 3. Western blotting analysis of BRD2, BRD3, and BRD4

proteins in RS4;11 cells treated with BET degraders 4, 6, and 9 and

BET inhibitor 8. RS4;11 cells were treated for 3 h with each individual

compound at indicated concentrations, and proteins were probed by

speciﬁc antibodies. GAPDH was used as the loading control.

previously reported BET degraders, 4 and 6, also eﬀectively

decrease the level of BRD2−4 proteins in the RS4;11 cell line

(Figure 3). Consistent with its testis-speciﬁc expression, BRDT

protein was not detected in the RS4;11 cells. We concluded

that 9 is a promising BET degrader for further optimization.

We next explored the length and the composition of the

linker in 9. Replacing the oxygen atom in 9 with an amino

group resulted in 10, whose cell growth inhibitory activity is 2

times better than that of 9. Conversion of the amide group

(-CO-NH-) in 9 to an ethylene group (-CH₂-CH₂-) generated

11, which has an IC₅₀value of 0.48 nM in inhibition of cell

growth in the RS4;11 cell line and is thus 10 times more potent

than 9. Similar conversion of the amide group in 10 to an

ethylene group yielded 12 which, with an IC₅₀value of 0.20 nM

in inhibition of RS4;11 cell growth, is 10 times more potent

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Table 3. Further Optimization of the Linker and Phthalimide Moiety

a

IC₅₀values were obtained from three independent experiments.

Table 4. Investigation of the Eﬀect of BET Protein and Cereblon Binding on Cellular Potencies of BET Degraders

a

IC₅₀values were obtained from three independent experiments.

mide in three potent BET degraders 18, 19, and 20. This eﬀort

resulted in 21, 22, and 23, which achieve IC₅₀values of 0.037

nM, 0.90 nM, and 0.051 nM, respectively, in inhibition of

RS4;11 cell growth. Hence, 21 and 23 are 2−3 times more

potent than 18 and 20. Interestingly, in contrast to 21 and 23,

compound 22 has an IC₅₀value of 0.9 nM in inhibition of

RS4;11 cell growth and is thus 2 times less potent than 19. To

further conﬁrm the signiﬁcance of the linker length, we

synthesized compounds 24 and 25 with one methylene or

ethylene group shorter than that in 23. Compounds 24 and 25

have IC₅₀values of 0.98 nM and 9.6 nM in inhibition of RS4;11

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cell growth, respectively, and are therefore 19 and 188 times

less potent than 23.

We further investigated the potency of compound 23 in

inducing BET protein degradation in the RS4;11 cells with a 24

h treatment, with compound 8 included as a control (Figure 5).

To further investigate the mechanism of action for this class

of BET degraders, we have designed two control compounds

based upon 23. It has been shown that installation of a methyl

group on the amino group in the lenalidomide moiety blocks

the binding of thalidomide analogues to cereblon.^25,29

Accordingly, we installed a methyl group on the amino group

in the lenalidomide moiety, which resulted in 26 (Table 4).

Compound 26 has an IC₅₀value of 35.1 nM in inhibition of

RS4;11 cell growth and is therefore >600 times less potent than

23, but the IC₅₀is similar to that obtained for the BET inhibitor

8. Furthermore, 26 fails to induce degradation of BRD2, BRD3,

and BRD4 proteins in the RS4;11 cell line, indicating that it

inhibits cell growth by acting not as a BET degrader but as a

BET inhibitor (Figure S1 in Supporting Information). We

synthesized compound 27 based upon a less potent BET

inhibitor 28, which binds to BRD2, BRD3, and BRD4 proteins

with aﬃnities of >100 times less potent than that of 8 (Table

1). Compound 27 has an IC₅₀value of >1000 nM and is thus

>10 000 and >400 times less potent than 23 in RS4;11 and

MOLM13 cell growth inhibition assays, respectively (Table 4).

These data indicate that in order to achieve potent cell growth

inhibition, a BET degrader must be able to bind to both BET

proteins and to the ubiquitin ligase complex, which is consistent

with their PROTAC design and the expected mechanism of

action.

Figure 5. Western blotting analysis of BRD2, BRD3, and BRD4

proteins, as well as c-Myc and PARP in RS4;11 cells treated with BET

degrader 23 and BET inhibitor 8. RS4;11 cells were treated for 24 h

with individual compounds at indicated concentrations, and proteins

were probed by speciﬁc antibodies. GAPDH was used as the loading

control.

Compound 23 is capable of eﬀectively reducing the level of

BRD2 and BRD3 proteins at concentrations as low as 0.1 nM

and the level of BRD4 protein at a concentration of 0.03 nM. In

comparison, the BET inhibitor 8 has no eﬀect on the level of

BRD2−4 proteins at all concentrations tested (30−1000 nM).

We examined the eﬀects of the BET degrader 23 and the

BET inhibitor 8 in the RS4;11 cell line on the level of c-Myc

(Figure 5), a protein known to be down-regulated by both BET

inhibitors and degraders.^25,30Consistent with its extremely high

potency in inducing BET protein degradation, 23 eﬀectively

down-regulates the level of c-Myc at concentrations as low as

0.1 nM. In comparison, the BET inhibitor 8 can also eﬀectively

down-regulate the level of c-Myc but at concentrations of 300−

1000 nM in the RS4;11 cell line. Hence, the BET degrader 23

is >1000 times more potent than the BET inhibitor 8 in down-

regulation of c-Myc protein in the RS4;11 cell line.

We examined the ability of four representative BET

degraders (21 and 23−25) to induce BET degradation in the

RS4;11 cell line, and the data are shown in Figure 4. All these

The mechanism of action of BET protein degradation by 23

was further investigated (Figure 6). Addition of the BET

inhibitor 8 eﬀectively blocks the degradation of BRD2, BRD3,

and BRD4 proteins induced by 23 (Figure 6A), further

conﬁrming that the degradation of BET proteins by 23 requires

its binding to BET proteins. Similarly, addition of lenalidomide

also eﬀectively blocks the degradation induced by 23 for all

three BET proteins (Figure 6B), clearly indicating that

degradation of BET proteins by 23 is cereblon-dependent.

The proteasome inhibitor MG-132 and the NEDD8-activating

enzyme (NAE) inhibitor MLN4924 also completely block the

degradation of BET proteins by 23 (Figure 6C), indicating that

BET protein degradation by 23 depends upon proteasome and

NAE. These mechanistic data constitute clear evidence that 23

is a bona ﬁde and highly potent BET degrader.

We employed ﬂow cytometry analysis to investigate the

ability of the BET degrader 23 and the BET inhibitor 8 to

induce cell cycle arrest and apoptosis in the RS4;11 and

MOLM-13 cell lines (Figure 7). Both compounds were found

to eﬀectively induce cell cycle arrest in a dose-dependent

manner in both cell lines, but they have very diﬀerent

potencies. While the BET inhibitor 8 is eﬀective at 30 nM in

the RS4;11 cell line in inducing cell cycle arrest, the BET

degrader 23 has a strong eﬀect at concentrations as low as 0.3

nM. In the MOLM-13 cell line, 8 is eﬀective at 30−100 nM in

Figure 4. Western blotting analysis of BRD2, BRD3, and BRD4

proteins in RS4;11 cells treated with compounds 21 and 23−25.

RS4;11 cells were treated for 3 h with individual compounds at

indicated concentrations, and proteins were probed by speciﬁc

antibodies. GAPDH was used as the loading control.

four compounds can eﬀectively and potently induce degrada-

tion of BET proteins in a dose-dependent manner after a 3 h

treatment, and their potencies in reducing the levels of BRD2−

4 proteins correlate well with their cellular potencies in

inhibition of cell growth in the RS4;11 cell line. At

concentrations as low as 10 nM, 25 eﬀectively decreases the

level of BRD3 and BRD4 proteins but is less potent and

eﬀective in decreasing in the level of BRD2 protein. Compound

24 is very eﬀective in reducing the level of BRD2 and BRD4 at

concentrations as low as 3 nM and the level of BRD3 at 1 nM.

The two most potent BET degraders, 21 and 23, are highly

eﬀective in decreasing the level of BRD2 and BRD4 proteins at

concentrations as low as 0.3 nM and decreasing the level of

BRD3 at 0.1 nM with a 3 h treatment. Therefore, 21 and 23 are

extremely potent degraders of BET proteins.

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the 6 h time point. Western blotting analysis was performed to

probe the level of BRD2, BRD3, and BRD4 proteins, as well as

c-Myc, caspase-3, and PARP proteins in the tumor tissue. Our

PD data (Figure 9) clearly show that a single dose of 23 was

highly eﬀective, dramatically reducing the level of BRD2,

BRD3, and BRD4 proteins in the RS4;11 tumor tissue, starting

from 1 h with the eﬀect persisting for >24 h. The level of c-Myc

was strongly down-regulated, with the eﬀect persisting for at

least 6 h. Robust cleavage of PARP and caspase-3 was observed,

starting from the 3 h time-point and with a peak eﬀect at the 6

h time point, indicating strong apoptosis induction by 23.

Therefore, our PD analysis clearly demonstrates that a single

dose of 23 is suﬃcient to induce near complete degradation of

BRD2, BRD3, and BRD4 proteins for >24 h, accompanied by

robust cleavage of PARP and caspase-3, and strong down-

regulation of c-Myc protein.

We analyzed concentrations of 23 in plasma and also in

RS4;11 tumors in the same mice used in the PD experiment

(Table 5). Our data showed that despite its relatively large size

(MW = 798.8), 23 can eﬀectively penetrate the RS4;11

xenograft tumor tissue and has a concentration of 166.3, 98.5,

and 35.8 ng/g in the tumor at 1, 3, and 6 h time-points,

respectively, exceeding the drug concentrations needed for

eﬀective BET degradation shown in our in vitro experiments

(Figures 4 and 5). The drug concentration, however, was

undetectable after 24 h. Together with the PD data, our PK

data indicate that a single dose of 23 at 5 mg/kg achieves a

suﬃcient exposure in the tumor tissue for eﬀective induction of

BET protein degradation for over 24 h.

We also evaluated the metabolism of compound 23 in mouse

liver microsomes. The major metabolite was proposed to be

mono- or dihydroxylated product occurring in the alkyl chain in

the linker. In addition, cleavage of the C(sp³)−C(sp²) bond

between the linker and cereblon binding ligand and N-

deethylation of the pyrazole moiety are other alternative

metabolic pathways (for detailed information, see Figure S5).

Figure 6. Western blotting analysis of BRD2, BRD3, and BRD4

proteins after a 2 h pretreatment with 8 (A), lenalidomide (B), or a

proteasome inhibitor MG-132 or a NEDD8-activating enzyme (NAE)

inhibitor MLN4924 (C), followed by a 3 h treatment with 23 at 1 nM

in RS4;11 cells.

CHEMISTRY

inducing cell cycle arrest while 23 is very eﬀective at 1−3 nM.

We also observed a sharp contrast in their ability to induce

apoptosis. While the BET degrader 23 induces robust apoptosis

in both RS4;11 and MOLM-13 cell lines at 3−10 nM

concentrations with a 24 h treatment, the BET inhibitor 8 is

ineﬀective in both cell lines at concentrations as high as 300

nM.

■

The synthesis of compounds 8 and 28 is outlined in Scheme 1.

Brieﬂy, 3-cyclopropyl-3-oxopropanenitrile (29) and ethyl-

hydrazine oxalate (30) were heated in EtOH to generate 31.

Compound 32 was prepared as described, and intermediate 33

was obtained from 32 in four steps.³¹Coupling of 31 with 33

followed by hydrolysis of the methyl ester produced the key

intermediate 34. Amidation of 34 with MeNH₂in the presence

of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-

pyridinium 3-oxide hexaﬂuorophosphate (HATU) and N,N-

diisopropylethylamine (DIPEA) at room temperature in

dimethylformamide (DMF) gave the target compound 8.

Compound 36 was formed by dehalogenation of 33 in the

presence of Pd/C and H₂, followed by hydrolysis. Acyl chloride

formation followed by amidation of 36 generated 28.

The synthesis of compound 9 is shown in Scheme 2.

Condensation of 3-hydroxyphthalic anhydride (37) with 3-

aminopiperidine-2,6-dione hydrochloride (38) aﬀorded the

intermediate 39. Alkylation of 39 with tert-butyl bromoacetate

generated 40, which was subjected to triﬂuoroacetic acid-

promoted ester hydrolysis. Amide condensation and Boc-

deprotection reactions aﬀorded 41. Condensation of 41 with

34 under the same conditions described for the synthesis of 8

gave compound 9.

We tested the antitumor activity of 23 in the RS4;11

xenograft model in mice, and the results are shown in Figure 8.

Dosing of the mice bearing RS4;11 xenograft tumors with 5

mg/kg of 23 intravenously every other day, three times a week

for 3 weeks, achieved rapid tumor regression with a maximum

of >90% regression observed. There was no animal weight loss

or other signs of toxicity in mice treated with compound 23,

and the animal weight gained in the vehicle control group of

mice was attributed largely to rapid tumor growth. Therefore,

the in vivo data ﬁrmly establish that 23 has highly eﬃcacious

antitumor activity at a well-tolerated dose-schedule in mice.

To gain a better understanding of the strong antitumor

activity of 23 and its mechanism of action in vivo, we

performed a pharmacodynamics (PD) analysis in SCID mice

bearing the RS4;11 xenograft tumors (Figure 9). In this

experiment, mice bearing one or two RS4;11 xenograft tumors

were administered a single dose of 23 at 5 mg/kg intravenously

and were sacriﬁced at 1, 3, 6, and 24 h time-points after the

treatment. Mice treated with vehicle control were sacriﬁced at

The synthetic route to compound 10 is shown in Scheme 3.

Compound 43 was obtained by treatment of 3-nitrophthalic

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Figure 7. Induction of cell cycle arrest and apoptosis by BET degrader 23 and BET inhibitor 8 in the RS4;11 and MOLM-13 cell lines by ﬂow

cytometry analysis.

anhydride (42) with benzyl bromide, followed by reduction of

the nitro group with stannous chloride dihydrate and alkylation

with tert-butyl bromoacetate. Removal of the benzyl groups of

43 followed by amide condensation resulted in 44. Compound

10 was prepared from 44 by the same procedure as was used

for the synthesis of 9.

Synthesis of compounds 19 and 22 is shown in Scheme 7.

Bromination of 57 with N-bromosuccinimide (NBS) and

benzoyl peroxide (BPO) gave 58, which was heated with 38

and triethylamine (TEA) in MeCN to generate 59.

Compounds 19 and 22 were prepared starting from 53 and

59, respectively, in the same way as 53 was converted to 18 and

20.

As shown in Scheme 4, compound 11 was synthesized

starting from the intermediate 39. Alkylation of the hydroxyl

group of 39 with 7-bromo-1-heptanol gave 46. Compound 47

was obtained by conversion of the hydroxyl group to an amine

group in 46 through three steps. Condensation of 47 with 34

using previously described methods aﬀorded compound 11.

Compounds 12−17 were synthesized according to the route

shown in Scheme 5. Compound 49 was prepared from 48 by

the strategy used in the preparation of 39. Compounds 50a−f

were formed by substitution reaction of 49 with diﬀerent length

of mono-Boc protected alkyl diamines. Boc-deprotection with

TFA in DCM aﬀorded the intermediates 51a−f, which were

subsequently condensed with 34 to generate compounds 12−

17.

The synthesis of compounds 18 and 20 is shown in Scheme

6. Sonogashira coupling of 53, which was prepared in a manner

similar to that used to produce 49, with diﬀerent terminal

alkyne-containing linker substrates gave 54a,b. Reduction of the

alkyne group in 54a,b resulted in 55a,b, whose Boc group was

removed in TFA/DCM to form 56a,b. Compounds 18 and 20

were obtained by condensation of 56a,b with 34 using the same

method as was used for 9.

Starting from 59, compounds 21, 23−25, and 27 were

prepared using the procedure outlined in Scheme 8, which is

similar to the procedure used in the preparation of 22 from 59.

Finally, the synthesis of compound 26 is outlined in Scheme

9. Methylation of 59 with MeI yielded 63. Compound 26 was

prepared from 63 by a process similar to that used for the

synthesis of 18 and 20 from 53.

CONCLUSION

■

Upon the basis of a novel, azacarbazole-containing potent BET

inhibitor and thalidomide/lenalidomide, we have designed a

new class of PROTAC small-molecule degraders of BET

proteins. Through extensive optimization of the linker length

and composition, we have obtained a number of highly potent

small-molecule BET protein degraders as exempliﬁed by

compound 23. Compound 23 eﬀectively induces degradation

of the BET proteins BRD2, BRD3, and BRD4 at concentrations

as low as 0.1−0.3 nM with a 3 h treatment in the RS4;11 acute

leukemia cell line and is capable of inducing degradation of

BRD4 protein at concentrations as low as 30 pM with a 24 h

treatment. Compound 23 achieves an IC₅₀value of 51 pM and

2.3 nM in inhibition of cell growth in the RS4;11 and MOLM-

H

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Figure 8. BET degrader 23 induces complete tumor regression in the xenograft model of human RS4;11 tumor cells with minimal body weight

change.

were reported in parts per million (ppm) downﬁeld from

13 acute leukemia cell lines, respectively. Our mechanistic

investigation ﬁrmly establishes that compound 23 is a bona ﬁde

PROTAC BET degrader, whose activity requires that 23 binds

to BET proteins and the cereblon/Cullin4A E3 ubiquitin ligase

complex. Compound 23 eﬀectively induces both cell cycle

arrest and apoptosis in RS4;11 and MOLM-13 acute leukemia

cell lines at subnanomolar to low nanomolar concentrations.

Signiﬁcantly, 23 achieves >90% tumor regression in the RS4;11

xenograft model in mice at a well-tolerated dose-schedule. Our

PD analysis showed that a single, intravenous dose of 23 is

capable of inducing profound degradation of BET proteins in

the tumor tissue with eﬀect persisting for >24 h, accompanied

by robust cleavage of PARP and caspase-3 and strong down-

regulation of c-Myc. In addition to its potent anticancer activity

against acute leukemia cells, our recent study also showed that

compound 23 is very potent and eﬃcacious against triple-

negative human breast cancer in vitro and in vivo.³²

Collectively, our data demonstrate that 23 is a highly potent,

eﬃcacious, and promising BET degrader and warrants further

evaluation as a potential new therapy for the treatment of

human acute leukemia and other types of human cancer.

tetramethylsilane (TMS). All ¹³C NMR spectra were reported in

1

ppm and obtained with H decoupling. In reported spectral data, the

format (δ) chemical shift (multiplicity, J values in Hz, integration) was

used with the following abbreviations: s = singlet, d = doublet, t =

triplet, q = quartet, m = multiplet. MS analyses were carried out with a

Waters UPLC−mass spectrometer. The ﬁnal compounds were all

puriﬁed by C18 reverse phase preparative HPLC column with solvent

A (0.1% TFA in H₂O) and solvent B (0.1% TFA in MeCN) as eluents.

The purity of all the ﬁnal compounds was conﬁrmed to be >95%

purity by UPLC−MS or UPLC.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-6-methoxy-N-methyl-9H-pyrimido[4,5-b]-

indole-2-carboxamide (8). Ethylhydrazine oxalate (30, 41.0 g, 0.27

mmol, 1.2 equiv) in 200 mL of EtOH was added to a stirred solution

of 3-cyclopropyl-3-oxopropanenitrile (29, 25.0 g, 0.23 mol, 1.0 equiv).

The resulting solution was heated to reﬂux for 12 h. After cooling to

room temperature, most of the solvent was evaporated. Workup was

performed with EtOAc and saturated brine solution. The combined

organic layer was dried over anhydrous Na₂SO₄. After ﬁltration and

concentration, the residue was puriﬁed by ﬂash column chromatog-

raphy with hexane/EtOAc to aﬀord the desired compound 31 as a

slightly yellow solid (22.6 g, 65% yield). ¹H NMR (400 MHz, CDCl₃)

5.07 (s, 1H), 3.82 (q, J = 7.2 Hz, 2H), 3.55 (s, 2H, NH₂), 1.78−1.71

(m, 1H), 1.28 (t, J = 7.2 Hz, 3H), 0.80−0.75 (m, 2H), 0.57−0.54 (m,

2H). UPLC−MS calculated for C₈H₁₄N₃[M + H]⁺: 152.12, found

152.09.

EXPERIMENTAL SECTION

■

Chemistry. General Experiment and Information. Unless

otherwise noted, all purchased reagents were used as received without

further puriﬁcation. ¹H NMR and ¹³C NMR spectra were recorded on

Compound 32, Prepared in Three Steps Following a

Published Method.³²Compound 32 (13.16 g, 40 mmol, 1.0

equiv) and ethyl cyanoformate (19.76 mL, 0.2 mol, 5.0 equiv) were

1

a Bruker Advance 400 or 300 MHz spectrometer. H NMR spectra

I

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1

late as a solid (10.61 g, 90% yield). H NMR (300 MHz, CD₃OD) δ

(ppm) 7.86 (s, 1H), 7.39 (s, 1H), 4.07 (s, 3H), 3.93 (s, 3H), 2.34 (s,

3H), 2.17 (s, 3H).

Methyl 7-(3,5-dimethylisoxazol-4-yl)-4-hydroxy-6-methoxy-9H-

pyrimido[4,5-b]indole-2-carboxylate (10.61 g, 28.8 mmol) and

POCl₃(200 mL) were stirred in a round-bottom ﬂask at 90 °C for

12 h, then cooled to room temperature, and the volatile components

were removed on a rotary evaporator. EtOAc (200 mL) was added,

and the pH was adjusted to 8 using aqueous NaOH solution. Filtration

of the mixture furnished compound 33 as a yellow solid (7.80 g, 70%

yield). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 7.81 (s, 1H), 7.55 (s,

1H), 3.92 (s, 3H), 3.90 (s, 3H), 2.33 (s, 3H), 2.12 (s, 3H). UPLC−MS

calculated for C₁₈H₁₆ClN₄O₄[M + H]⁺: 387.09, found 387.18.

Pd₂(dba)₃(183 mg, 0.2 mmol, 0.1 equiv), racemic BINAP (249 mg,

0.4 mmol, 0.2 equiv), and K₃PO₄(1.70 g, 8.0 mmol, 4.0 equiv) were

added sequentially to a solution of compound 33 (774 mg, 2.0 mmol,

1.0 equiv) in toluene (25 mL). The solution was purged and reﬁlled

with nitrogen three times before compound 31 (604 mg, 4.0 mmol,

2.0 equiv) was added. The solution was purged and reﬁlled with

nitrogen again. The resulting solution was heated to 110 °C and

stirred for 12 h. After cooling to room temperature, the solution was

ﬁltered through Celite and concentrated. The residue was puriﬁed by

ﬂash column chromatography with DCM/MeOH to aﬀord methyl 4-

((3-cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-dimethylisoxa-

zol-4-yl)-6-methoxy-9H-pyrimido[4,5-b]indole-2-carboxylate as a yel-

low solid (702 mg, 70% yield). ¹H NMR (400 MHz, DMSO-d₆) 12.15

(s, 1H), 12.05 (s, 1H), 8.06 (s, 1H), 7.56 (s, 1H), 7.22 (s, 1H), 3.99

(q, J = 7.2 Hz, 2H), 3.79 (s, 3H), 3.13 (s, 3H), 2.24 (s, 3H), 2.05 (s,

3H), 1.78−1.71 (m, 1H), 1.14 (t, J = 7.2 Hz, 3H), 0.80−0.75 (m, 2H),

0.57−0.54 (m, 2H).

Figure 9. Pharmacodynamic analysis of compound 23 in RS4;11

xenograft tumor tissue. SCID mice bearing RS4;11 tumors were

treated with a single intravenous dose of 23 at 5 mg/kg. Mice were

sacriﬁced at 1, 3, 6, and 24 h time-points after administration with

compound 23 or at 6 h with vehicle control, and tumors were

harvested from mice for Western blotting analysis of BRD2, BRD3,

BRD4, c-Myc, PARP and cleaved PARP (Cl PARP), and caspase-3 and

cleaved caspase-3 (Cl caspase-3). Actin was used as the loading

control. Two mice were used for each time-point with each mouse

bearing either one or two tumors.

LiOH (101 mg, 4.2 mmol, 3.0 equiv) was added to a solution of

methyl 4-((3-cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-6-methoxy-9H-pyrimido[4,5-b]indole-2-carboxy-

late (702 mg, 1.4 mmol, 1.0 equiv) in THF (20 mL). The solution was

stirred at room temperature for 12 h. After concentration, the residue

was puriﬁed by preparative HPLC to aﬀord the desired compound 34

as a yellow solid (545 mg, 80% yield).

added to a round-bottom ﬂask at room temperature. A 4.0 M

hydrogen chloride solution in dioxane (90 mL) was added, and the

reaction mixture was reﬂuxed at 82 °C for 12 h. The reaction was then

cooled to room temperature, and the solvents were removed on a

rotary evaporator. To this crude mixture, 10% NaOH aqueous solution

(120 mL) and EtOH (100 mL) were added, and the solution was

heated to reﬂux for 6 h. The volatile components were then removed

on a rotary evaporator, and the aqueous residue was acidiﬁed with 2 N

aqueous HCl. The product was allowed to precipitate at 0 °C.

Filtration of the mixture furnished pure 7-(3,5-dimethylisoxazol-4-yl)-

4-hydroxy-6-methoxy-9H-pyrimido[4,5-b]indole-2-carboxylic acid as a

yellow solid (11.34 g, 80% yield). UPLC−MS calculated for

C₁₇H₁₅N₄O₅[M + H]⁺: 355.10, found 355.45.

Concentrated sulfuric acid (15 mL) was added to a solution of 7-

(3,5-dimethylisoxazol-4-yl)-4-hydroxy-6-methoxy-9H-pyrimido[4,5-b]-

indole-2-carboxylic acid (11.34 g, 32 mmol) in EtOH (300 mL) in a

round-bottom ﬂask. The mixture was heated to reﬂux for 12 h. The

volatile components were removed on a rotary evaporator. Then 400

mL of EtOAc was added. The product was allowed to precipitate.

Filtration of the mixture furnished pure methyl 7-(3,5-dimethylisox-

azol-4-yl)-4-hydroxy-6-methoxy-9H-pyrimido[4,5-b]indole-2-carboxy-

HATU (27 mg, 0.07 mmol, 1.4 equiv), DIPEA (27 μL, 0.15 mmol,

3.0 equiv), and methylamine (2.0 M in THF 0.1 mL, 0.20 mmol, 4.0

equiv) were added sequentially to a stirred solution of compound 34

(24 mg, 0.05 mmol, 1.0 equiv) in DMF (2.0 mL). The solution was

stirred at room temperature for 2 h. Preparative HPLC aﬀorded the

1

pure product (8) as a slightly yellow solid (17 mg, 68% yield). H

NMR (400 MHz, DMSO-d₆) δ (ppm) 12.17 (s, 1H), 9.27 (s, 1H),

8.21 (s, 1H), 7.43 (s, 1H), 7.34 (s, 1H), 5.92 (s, 1H), 3.96 (q, J = 7.2

Hz, 2H), 3.81 (s, 3H), 2.81 (d, J = 4.8 Hz, 3H), 2.30 (s, 3H), 2.09 (s,

3H), 1.92−1.85 (m, 1H), 1.32 (t, J = 7.2 Hz, 3H), 0.87−0.82 (m, 2H),

0.64−0.60 (m, 2H). UPLC−MS calculated for C₂₆H₂₉N₈O₃[M + H]⁺:

501.24, found 501.22. Purity, 99.0%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-

dioxoisoindolin-4-yl)oxy)acetamido)butyl)-6-methoxy-9H-

pyrimido[4,5-b]indole-2-carboxamide (9). In a round-bottom

ﬂask, 3-hydroxyphthalic anhydride 37 (1.0 g, 6.09 mmol, 1.0 equiv)

and 3-aminoperidine-2,6-dione hydrochloride 38 (1.0 g, 6.09 mmol,

1.0 equiv) were mixed in toluene (50 mL). TEA (0.93 mL, 6.7 mmol,

a

Table 5. Analysis of Concentrations of Compound 23 in Plasma and RS4;11 Tumor Tissue

concn in plasma (ng/mL)

individual mice

concn in tumor (ng/g)

time-point (h)

mean

individual tumor

mean

SD

1

3

422

58

362

392

67.1

6.4

135.5

86

123

89

240.5

120.5

36.2

<1

166.3

98.5

35.8

<1

64.5

19.1

3.8

76.1

6

5.5

7.4

<1

39.4

<1

31.9

<1

24

<1

a

Mice bearing RS4;11 xenograft tumors were administered with a single dose of compound 23 at 5 mg/kg intravenously. Mice were sacriﬁced at 1, 3,

6, and 24 h time-points after administration with compound 23, and plasma and tumor tissue were collected for analysis. Two mice were used for

each time-point with each mouse bearing either one or two tumors (three tumors for each time point).

J

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a

Scheme 1. Compounds 8 and 28

a

Reaction conditions: (a) EtOH, reﬂux; (b) NCCOOEt, 4 N HCl in dioxane; (c) 10% NaOH (aq), EtOH, reﬂux; (d) conc H₂SO₄, MeOH, reﬂux;

(e) POCl₃, reﬂux; (f) Pd₂(dba)₃, BINAP, K₃PO₄, toluene, reﬂux; (g) LiOH, THF, rt; (h) NH₂Me, HATU, DIPEA, DMF, rt; (i) Pd/C, H₂, DMF, 60

°C; (j) LiOH, THF, rt; (k) 2 drops of DMF, SOCl₂, reﬂux; (l) methylamine hydrochloride, THF, rt.

a

Scheme 2. Compound 9

a

Reaction conditions: (a) TEA, toluene, reﬂux; (b) tert-butyl bromoacetate, KI, KHCO₃, DMF, 60 °C; (c) TFA, rt; (d) N-Boc-1,4-butanediamine,

HATU, DIPEA, DMF, rt; (e) TFA, DCM, rt; (f) HATU, DIPEA, DMF, rt.

1.1 equiv) was added. The resulting reaction mixture was heated to

reﬂux for 12 h with Dean−Stark trap equipment. After cooling to

ambient temperature, evaporation of most of the solvent aﬀorded a

crude product, which was puriﬁed by ﬂash column chromatography

with DCM/MeOH to obtain the desired product 39 as a slightly

was added dropwise. The resulting mixture was stirred at 60 °C for 12

h. After normal workup with EtOAc and saturated brine, the combined

organic layer was dried over Na₂SO₄. After ﬁltration and evaporation,

the residue was puriﬁed by ﬂash column chromatography with DCM/

MeOH to get the intermediate 40 as a white solid (1.7 g, 80% yield).

¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 11.13 (s, 1H), 7.80 (t, J =

8.0 Hz, 1H), 7.48 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 8.4 Hz, 1H), 5.13

(dd, J = 12.8 Hz, J = 5.2 Hz, 1H), 4.97 (s, 2H), 2.97−2.85 (m, 1H),

2.65−2.52 (m, 2H), 2.14−2.03 (m, 1H), 1.43 (s, 9H).

1

yellow solid (1.5 g, 90% yield). H NMR (400 MHz, DMSO-d₆) δ

(ppm) 11.16 (s, 1H), 11.08 (s, 1H), 7.65 (t, J = 7.6 Hz, 1H), 7.32 (d, J

= 7.2 Hz, 1H), 7.25 (d, J = 8.4 Hz, 1H), 5.07 (dd, J = 12.8 Hz, J = 5.2

Hz, 1H), 2.93−2.84 (m, 1H), 2.61−2.46 (m, 1H), 2.05−2.01 (m, 1H);

¹³C NMR (100 MHz, DMSO-d₆) δ (ppm) 173.28, 170.48, 167.50,

166.30, 155.99, 136.83, 133.60, 124.05, 114.82, 114.72, 49.11, 31.43,

22.51.

In a round-bottom ﬂask, compound 39 (1.5 g, 5.5 mmol, 1.0 equiv)

was dissolved in DMF (10 mL). KI (91 mg, 0.55 mmol, 0.1 equiv) and

KHCO₃(826 mg, 8.25 mmol, 1.5 equiv) were added to the stirred

solution. Then tert-butyl bromoacetate (0.98 mL, 6.6 mmol, 1.2 equiv)

The intermediate 40 was dissolved in TFA (8.0 mL). The reaction

mixture was stirred at room temperature for 2 h. After evaporation of

the solvent, the residue was freeze-dried on a lyophilizer to aﬀord a

white solid, which was used in the following steps without further

1

puriﬁcation. H NMR (400 MHz, DMSO-d₆) δ (ppm) 13.16 (s, 1H),

11.11 (s, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.48 (d, J = 7.2 Hz, 1H), 7.40

(d, J = 8.8 Hz, 1H), 5.11 (dd, J = 12.8 Hz, J = 5.2 Hz, 1H), 4.99 (s,

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a

Scheme 3. Compound 10

a

Reaction conditions: (a) TsOH·H₂O, BnOH, 100 °C; BnBr, NaHCO₃, DMF, 100 °C; (b) SnCl₂·2H₂O, EtOAc, 50 °C; (c) tert-butyl bromoacetate,

DIPEA, DMF, 90 °C; (d) Pd/C, H₂, EtOH, rt; (e) 38, pyridine, 110 °C; (f) TFA, rt; (g) N-Boc-1,4-butanediamine, HATU, DIPEA, DMF, rt; (h)

TFA, DCM, rt; (i) HATU, DIPEA, DMF, rt.

a

Scheme 4. Compound 11

a

Reaction conditions: (a) 7-bromoheptan-1-ol, NaHCO₃, KI, DMF, 80 °C; (b) MsCl, TEA, DCM; (c) NaN₃, DMF, 80 °C; (d) PPh₃, THF/H₂O,

rt; (e) HATU, DIPEA, DMF, rt.

1

2H), 2.95−2.86 (m, 1H), 2.63−2.48 (m, 2H), 2.08−2.03 (m, 1H); ¹³

C

solid (27 mg, 63% yield). H NMR (400 MHz, DMSO-d₆) δ (ppm)

12.21 (s, 1H), 11.10 (s, 1H), 9.30 (s, 1H), 8.23 (t, J = 6.0 Hz, 1H),

8.00 (t, J = 6.0 Hz, 1H), 7.81 (t, J = 7.6 Hz, 1H), 7.47 (d, J = 7.2 Hz,

1H), 7.39 (d, J = 8.4 Hz, 1H), 7.34 (s, 1H), 5.93 (s, 1H), 5.11 (dd, J =

12.8 Hz, J = 5.2 Hz, 1H), 4.78 (s, 2H), 3.95 (q, J = 7.2 Hz, 2H), 3.82

(s, 3H), 3.30−3.25 (m, 2H), 3.22−3.16 (m, 2H), 2.89−2.85 (m, 1H),

2.60−2.45 (m, 2H), 2.30 (s, 3H), 2.10 (s, 3H), 2.05−2.01 (m, 1H),

1.90−1.85 (m, 1H), 1.51−1.49 (m, 4H), 1.31 (t, J = 7.2 Hz, 3H),

0.86−0.81 (m, 2H), 0.63−0.60 (m, 2H). UPLC−MS calculated for

C₄₄H₄₆N₁₁O₉[M + H]⁺: 872.35, found 872.37. Purity, >99.0%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-

dimethylisoxazol-4-yl)-N-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-

1,3-dioxoisoindolin-4-yl)amino)acetamido)butyl)-6-methoxy-

9H-pyrimido[4,5-b]indole-2-carboxamide (10). In a round-

bottom ﬂask, 3-nitrophthalic anhydride 42 (5.79 g, 30 mmol, 1.0

equiv) and p-toluenesulfonic acid monohydrate (571 mg, 3 mmol, 0.1

equiv) were mixed in benzyl alcohol (20 mL). The mixture was heated

to 100 °C and stirred for 12 h. After cooling to room temperature,

benzyl bromide (7.1 mL, 45 mmol, 1.5 equiv), KI (498 mg, 3 mmol,

0.1 equiv), KHCO₃(9.0 g, 90 mmol, 3.0 equiv), and DMF (25 mL)

were added. The mixture was heated to 100 °C and stirred for 6 h.

After cooling to room temperature, the solvent was evaporated and the

NMR (100 MHz, DMSO-d₆) δ (ppm) 173.26, 170.38, 169.97, 167.21,

165.64, 155.61, 137.23, 133.73, 120.35, 116.80, 116.23, 65.48, 49.27,

31.42, 22.44. UPLC−MS calculated for C₁₅H₁₃N₂O₇[M + H]⁺:

333.07, found 333.17.

In a round-bottom ﬂask, the intermediate obtained from the

previous step (99.7 mg, 0.3 mmol, 1.0 equiv) was dissolved in

anhydrous DMF (2 mL). N-Boc-1,4-butanediamine (68 mg, 0.36

mmol, 1.2 equiv), HATU (137 mg, 0.36 mmol, 1.2 equiv), and DIPEA

(157 μL, 0.9 mmol, 3.0 equiv) were added sequentially. The reaction

mixture was stirred at room temperature for 2 h and then puriﬁed by

HPLC to get the product as a slightly yellow solid (128 mg, 85% yield)

which was dissolved in DCM (2.0 mL) and TFA (1.0 mL). After

stirring for 1 h, the solvent was evaporated and the residue 41 was

used as a stored solution (0.1 M in DMF) in the next step without

further puriﬁcation.

HATU (27 mg, 0.07 mmol, 1.4 equiv), DIPEA (27 μL, 0.15 mmol,

3.0 equiv), and the amine intermediate 41 (0.1 M in DMF 0.7 mL,

0.07 mmol, 1.4 equiv) were added sequentially to a stirred solution of

compound 34 (24 mg, 0.05 mmol, 1.0 equiv) in DMF (2.0 mL). The

solution was then stirred at room temperature for 2 h. Preparative

HPLC puriﬁcation aﬀorded the pure product (9) as a slightly yellow

L

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Scheme 5. Compounds 12−17

a

Reaction conditions: (a) NaOAc, AcOH, reﬂux; (b) DIPEA, DMF, 80 °C; (c) TFA, DCM, rt; (d) HATU, DIPEA, DMF, rt.

a

Scheme 6. Compounds 18 and 20

a

Reaction conditions: (a) NaOAc, AcOH; (b) tert-butyl pent-4-yn-1-ylcarbamate or tert-butyl hex-5-yn-1-ylcarbamate, Pd(PPh₃)₂Cl₂, CuI, DMF/

TEA, 80 °C; (c) Pd/C, H₂, EtOH, rt; (d) TFA, DCM, rt; (e) HATU, DIPEA, DMF, rt.

mixture was then poured into H₂O (300 mL). The solution was

extracted with EtOAc. The combined organic layers were washed with

brine and dried over anhydrous Na₂SO₄. After ﬁltration and

evaporation, the crude residue was puriﬁed by ﬂash column

chromatography with hexane/EtOAc to give the intermediate dibenzyl

3-nitrophthalate as a slightly yellow solid (9.4 g, 80% yield).

M

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Scheme 7. Compounds 19 and 22

a

Reaction conditions: (a) NBS, BPO, benzene, reﬂux; (b) TEA, MeCN, reﬂux; (c) tert-butyl (2-(prop-2-yn-1-yloxy)ethyl)carbamate, Pd(PPh₃)₂Cl₂,

CuI, DMF/TEA, 80 °C; (d) Pd/C, H₂, EtOH, rt; (e) TFA, DCM, rt; (f) HATU, DIPEA, DMF, rt.

a

Scheme 8. Compounds 21, 23−25, and 27

a

Reaction conditions: (a) tert-butyl prop-2-yn-1-ylcarbamate or tert-butyl but-3-yn-1-ylcarbamate or tert-butyl pent-4-yn-1-ylcarbamate or tert-butyl

hex-5-yn-1-ylcarbamate, Pd(PPh₃)₂Cl₂, CuI, DMF/TEA, 80 °C; (b) Pd/C, H₂, EtOH, rt; (c) TFA, DCM, rt; (d) HATU, DIPEA, DMF, rt.

a

Scheme 9. Compound 26

a

Reaction conditions: (a) MeI, K₂CO₃, DMF, 60 °C; (b) tert-butyl pent-4-yn-1-ylcarbamate, Pd(PPh₃)₂Cl₂, CuI, DMF/TEA, 80 °C; (c) Pd/C, H₂,

EtOH, rt; (d) TFA, DCM, rt; (e) HATU, DIPEA, DMF, rt.

In a round-bottom ﬂask, dibenzyl 3-nitrophthalate (9.4 g, 24 mmol,

1.0 equiv) was dissolved in EtOAc (100 mL). Then stannous chloride

dihydrate (11.3 g, 50 mmol, 2.08 equiv) was added portionwise to the

reaction mixture. The resulting reaction mixture was heated to 50 °C

and stirred for 12 h. Then NaOH (aq) was added to the reaction

mixture to quench the reaction. The reaction mixture was ﬁltered

through Celite and washed with EtOAc. The ﬁltrate was extracted with

EtOAc and brine. The combined organic layer was dried over

anhydrous Na₂SO₄. After ﬁltration and evaporation, the crude residue

was puriﬁed by ﬂash column chromatography with hexane/EtOAc to

give dibenzyl 3-aminophthalate as a slightly yellow solid (7.8 g, 90%

yield). H NMR (400 MHz, CDCl₃) δ (ppm) 7.45−7.36 (m, 10H),

7.22 (t, J = 8.4 Hz, 1H), 6.96 (d, J = 7.2 Hz, 1H), 6.76 (d, J = 7.6 Hz,

1H), 5.36 (s, 2H, NH₂), 5.26 (s, 2H), 5.10 (s, 2H).

1

In a round-bottom ﬂask, dibenzyl 3-aminophthalate (2.0 g, 5.54

mmol, 1.0 equiv) and KI (100 mg, 0.56 mmol, 0.1 equiv) were

N

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combined with anhydrous DMF (10 mL). tert-Butyl bromoacetate (2.4

mL, 16.6 mmol, 3.0 equiv) and DIPEA (4.8 mL, 27.7 mmol, 5.0 equiv)

were added to the reaction mixture which was then heated to 90 °C

and stirred for 12 h. After cooling to room temperature, most of the

solvent was evaporated and the residue was puriﬁed by column

chromatography with hexane/EtOAc to give compound 43 as a

slightly yellow solid (1.05 g, 40% yield). ¹H NMR (400 MHz, CDCl₃)

δ (ppm) 7.40−7.25 (m, 11H), 6.87 (d, J = 7.2 Hz, 1H), 6.65 (d, J =

8.4 Hz, 1H), 5.20 (s, 2H), 4.95 (s, 2H), 3.88 (d, J = 4.8 Hz, 2H), 1.52

(s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 169.16, 168.86,

167.61, 148.44, 135.78, 135.66, 135.42, 132.82, 128.72, 128.62, 128.58,

128.40, 128.22, 128.16, 116.54, 113.89, 111.32, 82.38, 67.32, 66.97,

46.07, 28.27.

In a round-bottom ﬂask, compound 43 (1.0 g, 2.1 mmol) was

dissolved in EtOH (20 mL). 100 mg of Pd/C (10 wt %) was added

under N₂. The ﬂask was purged and reﬁlled with H₂three times. Then

the reaction mixture was stirred at room temperature under 1 atm of

H₂. Once the starting material disappeared, as judged by TLC, the

mixture was ﬁltered through Celite and washed with EtOH. After

evaporation of the solvent, 3-aminopiperidine-2,6-dione hydrochloride

38 (380 mg, 2.31 mmol, 1.1 equiv) and pyridine (20 mL) were added.

The reaction mixture was heated to 110 °C and stirred overnight. After

cooling to room temperature, the solvent was evaporated as much as

possible and the residue was poured into H₂O. After extraction three

times with EtOAc, the combined organic layer was washed with brine

and dried over anhydrous Na₂SO₄. After ﬁltration and evaporation, the

crude residue was puriﬁed by ﬂash column chromatography with

DCM/MeOH to give compound 44 as a yellow solid (325 mg, 40%

yield).

and concentrated. The residue was puriﬁed by preparative HPLC to

aﬀord 46 as a colorless solid (589 mg, 76% yield). H NMR (400

1

MHz, DMSO-d₆) δ (ppm) 11.10 (s, 1H), 7.80 (t, J = 7.80 Hz, 1H),

7.51 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.09 (dd, J = 12.8

Hz, J = 5.2 Hz, 1H), 4.20 (t, J = 6.4 Hz, 3H), 3.39 (t, J = 6.8 Hz, 2H),

2.90−2.86 (m, 1H), 2.62−2.50 (m, 2H), 2.08−2.02 (m, 1H), 1.78−

1.72 (m, 2H), 1.48−1.29 (m, 8H); ¹³C NMR (100 MHz, DMSO-d₆) δ

(ppm) 173.24, 170.42, 167.32, 165.77, 156.50, 137.47, 133.72, 120.23,

116.69, 115.57, 69.27, 61.16, 49.22, 32.94, 31.43, 29.07, 28.89, 25.94,

25.82, 22.48. UPLC−MS calculated for C₂₀H₂₅N₂O₆[M + H]⁺:

389.17, found 389.18.

MsCl (0.14 mL, 1.70 mmol, 1.2 equiv) and TEA (0.30 mL, 2.13

mmol, 1.5 equiv) were added sequentially to a solution of compound

46 (589 mg, 1.42 mmol, 1.0 equiv) in DCM (10 mL) at 0 °C. The

resulting solution was stirred at 0 °C for 2 h. Then the solvent was

evaporated to aﬀord the crude residue. Sodium azide (277 mg, 4.25

mmol, 3.0 equiv) was added to the above residue in DMF (6 mL). The

solution was stirred at 70 °C for 2 h and then ﬁltered through Celite.

The residue was puriﬁed by preparative HPLC to aﬀord the desired

product as a white solid (386 mg, 66% yield in two steps). 10% Pd/C

(30 mg) was added to the above compound in EtOH (10 mL). The

ﬂask was purged and reﬁlled with H₂three times. The solution was

stirred at room temperature under H₂for 12 h. Filtration through

Celite removed the Pd/C, and then the solution was concentrated and

the residue was puriﬁed by preparative HPLC to aﬀord the desired

compound 47 as a colorless oil (72 mg, 20% yield).

HATU (27 mg, 0.07 mmol, 1.4 equiv), DIPEA (27 μL, 0.15 mmol,

3.0 equiv), and the amine 47 (27.1 mg, 0.07 mmol, 1.4 equiv) were

added sequentially to a stirred solution of the intermediate 34 (24 mg,

0.05 mmol, 1.0 equiv) in DMF (2.0 mL). The solution was stirred at

room temperature for 2 h. Preparative HPLC puriﬁcation aﬀorded the

TFA (2.0 mL) was added to compound 44, obtained as described

above. The intermediate (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoin-

dolin-4-yl)glycine was obtained by evaporation of the solvent without

1

pure product 11 as a slightly yellow solid (19 mg, 45% yield). H

1

NMR (400 MHz, DMSO-d₆) δ (ppm) 12.30 (s, 1H), 11.08 (s, 1H),

9.28 (s, 1H), 8.16 (t, J = 5.6 Hz, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.52 (s,

1H), 7.51 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.34 (s, 1H),

5.94 (s, 1H), 5.07 (dd, J = 12.8 Hz, J = 5.6 Hz, 1H), 4.21 (t, J = 6.4 Hz,

1H), 3.94 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.29−3.24 (m, 2H), 2.91−

2.82 (m, 1H), 2.59−2.44 (m, 2H), 2.30 (s, 3H), 2.10 (s, 3H), 2.05−

1.99 (m, 1H), 1.91−1.84 (m, 1H), 1.81−1.74 (m, 2H), 1.53−1.47 (m,

4H), 1.41−1.30 (m, 7H), 0.86−0.81 (m, 2H), 0.64−0.60 (m, 2H).

UPLC−MS calculated for C₄₅H₄₉N₁₀O₈[M + H]⁺: 857.37, found

857.35. Purity, 98.2%.

further puriﬁcation. H NMR (400 MHz, DMSO-d₆) δ (ppm) 12.91

(s, 1H, COOH), 11.10 (s, 1H, NH), 7.59 (t, J = 7.6 Hz, 1H), 7.08 (d, J

= 6.8 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.86 (t, J = 5.6 Hz, 1H, NH),

5.08 (dd, J = 13.2 Hz, J = 5.6 Hz, 1H), 4.12 (d, J = 5.2 Hz, 2H), 2.94−

2.85 (m, 1H), 2.63−2.49 (m, 2H), 2.09−2.07 (m, 1H); ¹³C NMR

(100 MHz, DMSO-d₆) δ (ppm) 173.28, 171.90, 170.52, 169.26,

167.75, 146.28, 136.60, 132.48, 118.18, 111.54, 110.11, 60.22, 49.07,

31.46, 22.61. UPLC−MS calculated for C₁₅H₁₄N₃O₆[M + 1]⁺: 332.09,

found 332.00.

Following the procedure for the synthesis of compound 41,

compound 45 (250 mg, 0.76 mmol) was synthesized from the

intermediate (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-

glycine.

General Procedure for Synthesis of Compounds 12−17. In a

round-bottom ﬂask, 3-ﬂuorophthalic anhydride 48 (6.64 g, 40 mmol,

1.0 equiv), 3-aminopiperidine-2,6-dione hydrochloride 38 (6.58 g, 40

mmol, 1.0 equiv), and sodium acetate (3.94 g, 48 mmol, 1.2 equiv)

were mixed in AcOH (120 mL). The resulting reaction mixture was

heated to reﬂux at 140 °C for 12 h. After cooling to room temperature,

most of the AcOH was evaporated and the residue was puriﬁed by

ﬂash column chromatography with DCM/MeOH to obtain

compound 49 as a slightly yellow solid (9.7 g, 88% yield). UPLC−

HATU (27 mg, 0.07 mmol, 1.4 equiv), DIPEA (27 μL, 0.15 mmol,

3.0 equiv), and compound 45 (28 mg, 0.07 mmol, 1.4 equiv) were

added sequentially to a stirred solution of compound 34 (24 mg, 0.05

mmol, 1.0 equiv) in DMF (2.0 mL). The solution was stirred at room

temperature for 2 h. Preparative HPLC puriﬁcation aﬀorded the pure

1

product (10) as a slightly yellow solid (32 mg, 75% yield). H NMR

1

MS calculated for C₁₃H₁₀FN₂O₄[M + H]⁺: 277.06, found 277.02. H

(400 MHz, DMSO-d₆) δ (ppm) 12.32 (s, 1H), 11.09 (s, 1H), 9.41 (s,

1H), 8.26 (t, J = 5.6 Hz, 1H), 8.19 (t, J = 5.6 Hz, 1H), 7.58 (t, J = 8.0

Hz, 1H), 7.48 (s, 1H), 7.41 (s, 1H), 7.03 (d, J = 6.8 Hz, 1H), 6.87 (d, J

= 8.8 Hz, 1H), 5.94 (s, 1H), 5.07 (dd, J = 12.8 Hz, J = 5.6 Hz, 1H),

3.97 (q, J = 7.2 Hz, 2H), 3.94 (s, 2H), 3.82 (s, 3H), 3.29−3.26 (m,

2H), 3.15−3.12 (m, 2H), 2.93−2.84 (m, 1H), 2.61−2.45 (m, 2H),

2.30 (s, 3H), 2.10 (s, 3H), 2.05−2.01 (m, 1H), 1.92−1.85 (m, 1H),

1.51−1.45 (m, 4H), 1.31 (t, J = 7.2 Hz, 3H), 0.87−0.82 (m, 2H),

0.64−0.60 (m, 2H). UPLC−MS calculated for C₄₄H₄₇N₁₂O₈[M +

H]⁺: 871.36, found 871.39. Purity, 91.4%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(7-((2-(2,6-dioxopiperidin-3-yl)-1,3-di-

oxoisoindolin-4-yl)oxy)heptyl)-6-methoxy-9H-pyrimido[4,5-b]-

indole-2-carboxamide (11). KI (33 mg, 0.2 mmol, 0.1 equiv),

NaHCO₃(336 mg, 4.0 mmol, 2.0 equiv), and 7-bromo-1-heptanol

(468 mg, 2.4 mmol, 1.2 equiv) were added sequentially to a solution of

compound 39 (548 mg, 2.0 mmol, 1.0 equiv) in DMF (10 mL). The

resulting solution was heated to 60 °C and stirred for 12 h. After

cooling to room temperature, the solution was ﬁltered through Celite

NMR (400 MHz, DMSO-d₆) δ (ppm) 11.15 (s, 1H), 7.98−7.93 (m,

1H), 7.80−7.72 (m, 2H), 5.17 (dd, J = 13.2 Hz, J = 5.2 Hz, 1H), 2.95−

2.86 (m, 1H), 2.64−2.47 (m, 2H), 2.10−2.06 (m, 1H).

Mono-Boc protected alkyl diamines (1.13 mmol, 1.1 equiv) of

diﬀerent lengths were added to a stirred solution of compound 49

(285 mg, 1.03 mmol, 1.0 equiv) in DMF (6.0 mL) and DIPEA (0.36

mL, 2.06 mmol, 2.0 equiv). The reaction mixture was stirred at 90 °C

for 12 h. Then the mixture was cooled to room temperature, poured

into H₂O, and extracted twice with EtOAc. The combined organic

layer was washed with brine, dried over anhydrous Na₂SO₄. After

ﬁltration and evaporation, the crude residue was puriﬁed by

preparative HPLC to give the intermediate (50a−f) which was

dissolved in DCM (4.0 mL) and TFA (2.0 mL). After stirring for 1 h,

the solvent was evaporated to give the crude product 51a−f, which

was used in the next step without further puriﬁcation.

HATU (27 mg, 0.07 mmol, 1.4 equiv), DIPEA (27 μL, 0.15 mmol,

3.0 equiv), and the amine intermediate 51a−f (0.07 mmol, 1.4 equiv)

were added sequentially to a stirred solution of compound 34 (24 mg,

O

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Journal of Medicinal Chemistry

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0.05 mmol, 1.0 equiv) in DMF (2.0 mL). The solution was stirred at

room temperature for 2 h. Preparative HPLC puriﬁcation aﬀorded the

pure product 12−17.

7.55−7.48 (m, 1H), 7.46 (s, 1H), 7.33−7.27 (m, 1H), 7.20 (d, J = 8.6

Hz, 1H), 7.01 (d, J = 7.1 Hz, 1H), 6.16 (s, 1H), 5.05−4.95 (m, 1H),

4.20 (q, J = 7.2 Hz, 2H), 3.87 (s, 3H), 3.72−3.70 (m, 2H), 3.66−3.62

(m, 2H), 2.83−2.73 (m, 1H), 2.71−2.58 (m, 2H), 2.33 (s, 3H), 2.16

(s, 3H), 1.99−1.97 (m, 1H), 1.46 (t, J = 7.2 Hz, 3H), 1.11−1.02 (m,

2H), 0.83−0.79 (m, 2H). UPLC−MS (ESI⁺): m/z calculated for

C₄₀H₃₉N₁₁O₇[M + H]⁺: 786.30, found 786.18. UPLC analysis (10

min from 10% to 100% (MeCN/H₂O containing 0.1% CF₃CO₂H)):

retention time, 4.37 min; peak area, 97.4%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(6-(2-(2,6-dioxopiperidin-3-yl)-1,3-di-

oxoisoindolin-4-yl)hexyl)-6-methoxy-9H-pyrimido[4,5-b]-

indole-2-carboxamide (18). In a round-bottom ﬂask, 3-bromoph-

thalic anhydride 52 (2.27 g, 10.0 mmol, 1.0 equiv), 3-aminopiperidine-

2,6-dione hydrochloride 38 (1.81 g, 11 mmol, 1.1 equiv), and sodium

acetate (0.98 g, 12 mmol, 1.2 equiv) were mixed in acetic acid (30

mL). The resulting reaction mixture was heated to reﬂux at 140 °C for

12 h. After cooling to room temperature, most of the AcOH was

evaporated and the residue was puriﬁed by ﬂash column

chromatography with DCM/MeOH to give compound 53 as a purple

solid (2.70 g, 80% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 11.15 (s,

1H), 8.06 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 7.2 Hz, 1H), 7.78 (t, J = 7.6

Hz, 1H), 5.18 (dd, J = 12.8 Hz, J = 5.6 Hz, 1H), 2.95−2.86 (m, 1H),

2.65−2.48 (m, 2H), 2.11−2.05 (m, 1H); ¹³C NMR (100 MHz,

DMSO-d₆) δ 173.21, 170.18, 166.07, 165.65, 139.66, 136.73, 134.15,

129.20, 123.31, 118.11, 49.65, 31.39, 22.30. UPLC−MS calculated for

C₁₃H₁₀BrN₂O₄[M + H]⁺: 336.98, found 336.95.

In a round-bottom ﬂask, compound 53 (674 mg, 2.0 mmol, 1.0

equiv) and tert-butyl hex-5-yn-1-ylcarbamate (934 mg, 4.0 mmol, 2.0

equiv) were added to a solution of CuI (76 mg, 0.4 mmol, 0.2 equiv)

and Pd(PPh₃)₂Cl₂(140 mg, 0.2 mmol, 0.1 equiv) in DMF (10 mL).

The solution was purged and reﬁlled with nitrogen three times. Then

trimethylamine (5.0 mL) was added. The solution was purged and

reﬁlled with nitrogen again. The mixture was stirred at 70 °C for 3 h

under Ar. The solution was cooled to room temperature and ﬁltered

through Celite. The residue was puriﬁed by ﬂash column

chromatography to yield compound 54b as a slightly yellow solid

(653 mg, 72% yield). UPLC−MS calculated for C₂₄H₂₇N₃NaO₆[M +

Na]⁺= 476.18, found 476.18.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-

dimethylisoxazol-4-yl)-N-(7-((2-(2,6-dioxopiperidin-3-yl)-1,3-

dioxoisoindolin-4-yl)amino)heptyl)-6-methoxy-9H-pyrimido-

1

[4,5-b]indole-2-carboxamide (12). H NMR (400 MHz, DMSO-

d₆) δ (ppm) 12.30 (s, 1H), 11.08 (s, 1H), 9.31 (s, 1H), 8.17 (s, 1H),

7.58−7.54 (m, 2H), 7.39 (s, 1H), 7.08 (d, J = 8.4 Hz, 1H), 7.00 (d, J =

6.8 Hz, 1H), 6.52 (s, 1H), 5.95 (s, 1H), 5.04 (dd, J = 12.8 Hz, J = 5.6

Hz, 1H), 3.95 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.31−3.25 (m, 4H),

2.92−2.83 (m, 1H), 2.59−2.49 (m, 2H), 2.30 (s, 3H), 2.10 (s, 3H),

2.05−2.00 (m, 1H), 1.90−1.85 (m, 1H), 1.61−1.56 (m, 2H), 1.54−

1.49 (m, 2H), 1.45−1.28 (m, 6H), 1.30 (t, J = 7.2 Hz, 3H), 0.87−0.82

(m, 2H), 0.65−0.61 (m, 2H). UPLC−MS calculated for C₄₅H₅₀N₁₁O₇

[M + H]⁺: 856.39, found 856.39. Purity, 97.9%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-di-

oxoisoindolin-4-yl)amino)hexyl)-6-methoxy-9H-pyrimido[4,5-

b]indole-2-carboxamide (13). ¹H NMR (400 MHz, methanol-d₄) δ

7.54 (s, 1H), 7.46 (s, 1H), 7.39−7.36 (m, 1H), 7.02 (d, J = 7.4 Hz,

1H), 6.93 (d, J = 7.1 Hz, 1H), 5.49 (s, 1H), 5.05−4.98 (m, 1H), 4.24−

4.17 (q, J = 7.3 Hz, 2H), 3.98 (s, 2H), 3.89 (s, 3H), 3.37−3.22 (m,

6H), 3.00 (s, 2H), 2.88−2.84 (m, 2H), 2.68−2.64 (m, 2H), 2.34 (s,

3H), 2.17 (s, 3H), 1.98−1.91 (m, 1H), 1.73−1.68 (m, 2H), 1.48 (t, J =

7.3 Hz, 3H), 1.15−1.05 (m, 2H), 0.89−0.81 (m, 2H). UPLC−MS

calculated for C₄₄H₄₇N₁₁O₇[M + H]⁺: 842.37, found 842.36. UPLC

analysis (10 min from 10% to 100% (MeCN/H₂O containing 0.1%

CF₃CO₂H)): retention time, 5.21 min; peak area, 98.5%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(5-((2-(2,6-dioxopiperidin-3-yl)-1,3-di-

oxoisoindolin-4-yl)amino)pentyl)-6-methoxy-9H-pyrimido-

1

[4,5-b]indole-2-carboxamide (14). H NMR (400 MHz, MeOD-

d₄) δ 7.51−7.42 (m, 2H), 7.38 (s, 1H), 6.98 (d, J = 8.9 Hz, 1H), 6.88

(d, J = 7.0 Hz, 1H), 6.08 (s, 1H), 4.93−4.97 (m, 1H), 4.15 (q, J = 7.0

Hz, 2H), 3.88 (s, 3H), 3.52−3.48 (m, 2H), 3.35 (s, 4H), 2.74−2.53

(m, 2H), 2.34 (s, 3H), 2.17 (s, 3H), 2.05−1.93 (m, 4H), 1.78−1.64

(m, 2H), 1.42 (t, J = 7.0 Hz, 3H), 1.40−1.34 (m, 1H), 1.08−0.96 (m,

2H), 0.80−0.73 (m, 2H). UPLC−MS calculated for C₄₃H₄₅N₁₁O₇[M

+ H]⁺: 828.35, found 828.35. UPLC analysis (10 min from 10% to

100% (MeCN/H₂O containing 0.1% CF₃CO₂H)): retention time,

4.80 min; peak area, 99.4%.

10% Pd/C (60 mg) was added to a solution of compound 54b (653

mg, 1.44 mmol, 1.0 equiv) in EtOH (10 mL) under N₂. The solution

was purged and reﬁlled with hydrogen three times. The solution was

stirred at room temperature for 12 h. After ﬁltration, the solvent was

evaporated to give the crude residue 55b, which was dissolved in DCM

(4.0 mL) and TFA (2.0 mL). The reaction mixture was stirred at room

temperature for 1 h. Preparative HPLC puriﬁcation aﬀorded

compound 56b as a colorless oil (411 mg, 80% yield in two steps).

HATU (27 mg, 0.07 mmol, 1.4 equiv), DIPEA (27 μL, 0.15 mmol,

3.0 equiv), and 56b (25 mg, 0.07 mmol, 1.4 equiv) were added

sequentially to a stirred solution of compound 34 (24 mg, 0.05 mmol,

1.0 equiv) in DMF (2.0 mL). The solution was stirred at room

temperature for 2 h. Preparative HPLC puriﬁcation aﬀorded the pure

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-di-

oxoisoindolin-4-yl)amino)butyl)-6-methoxy-9H-pyrimido[4,5-

1

b]indole-2-carboxamide (15). H NMR (400 MHz, DMSO-d₆) δ

(ppm) 12.29 (s, 1H), 11.08 (s, 1H), 9.36 (s, 1H), 8.31 (t, J = 5.6 Hz,

1H), 7.56 (t, J = 7.2 Hz, 1H), 7.49 (s, 1H), 7.40 (s, 1H), 7.12 (d, J =

8.4 Hz, 1H), 7.01 (d, J = 6.8 Hz, 1H), 6.58 (s, 1H), 5.96 (s, 1H), 5.05

(dd, J = 12.8 Hz, J = 5.2 Hz, 1H), 3.97 (q, J = 7.2 Hz, 2H), 3.83 (s,

3H), 3.40−3.30 (m, 4H), 2.92−2.83 (m, 1H), 2.60−2.49 (m, 2H),

2.30 (s, 3H), 2.10 (s, 3H), 2.07−2.01 (m, 1H), 1.91−1.84 (m, 1H),

1.65−1.55 (m, 4H), 1.31 (t, J = 7.2 Hz, 3H), 0.86−0.81 (m, 2H),

0.64−0.60 (m, 2H). UPLC−MS calculated for C₄₂H₄₄N₁₁O₇[M +

H]⁺: 814.34, found 814.37. Purity, 89.9%.

1

product 18 as a slightly yellow solid (29 mg, 70% yield). H NMR

(400 MHz, DMSO-d₆) δ (ppm) 12.25 (s, 1H), 11.10 (s, 1H), 9.29 (s,

1H), 8.15 (t, J = 5.6 Hz, 1H), 7.78−7.69 (m, 3H), 7.58 (s, 1H), 7.37

(s, 1H), 5.94 (s, 1H), 5.12 (dd, J = 13.2 Hz, J = 5.2 Hz, 1H), 3.94 (q, J

= 7.2 Hz, 2H), 3.83 (s, 3H), 3.28−3.23 (m, 2H), 3.05 (t, J = 8.0 Hz,

2H), 2.93−2.83 (m, 1H), 2.61−2.49 (m, 2H), 2.30 (s, 3H), 2.10 (s,

3H), 2.07−2.03 (m, 1H), 1.89−1.82 (m, 1H), 1.66−1.60 (m, 2H),

1.53−1.47 (m, 2H), 1.36−1.27 (m, 7H), 0.84−0.79 (m, 2H), 0.63−

0.59 (m, 2H). UPLC−MS calculated for C₄₄H₄₇N₁₀O₇[M + H]⁺:

827.36, found 827.28. Purity, 98.7%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-di-

oxoisoindolin-4-yl)amino)propyl)-6-methoxy-9H-pyrimido-

1

[4,5-b]indole-2-carboxamide (16). H NMR (400 MHz, MeOD-

d₄) δ 7.55−7.49 (m, 2H), 7.39 (s, 1H), 7.04 (d, J = 8.8 Hz, 1H), 6.98

(d, J = 7.1 Hz, 1H), 4.89−4.81 (m, 1H), 4.18 (q, J = 7.3 Hz, 2H), 3.89

(s, 3H), 3.70−3.56 (m, 2H), 3.52−3.42 (m, 2H), 3.35 (d, J = 1.4 Hz,

2H), 2.34 (s, 3H), 2.17 (s, 3H), 2.05−2.01 (m, 4H), 1.88−1.79 (m,

1H), 1.44 (t, J = 7.3 Hz, 3H), 1.09−1.01 (m, 2H), 0.82−0.73 (m, 2H).

UPLC−MS calculated for C₄₁H₄₁N₁₁O₇[M + H]⁺: 800.32, found

800.23. UPLC analysis (10 min from 10% to 100% (MeCN/H₂O

containing 0.1% CF₃CO₂H)): retention time, 4.43 min; peak area,

98.0%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-di-

oxoisoindolin-4-yl)amino)ethyl)-6-methoxy-9H-pyrimido[4,5-

b]indole-2-carboxamide (17). ¹H NMR (400 MHz, methanol-d₄) δ

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(2-(3-(2-(2,6-dioxopiperidin-3-yl)-1,3-

dioxoisoindolin-4-yl)propoxy)ethyl)-6-methoxy-9H-pyrimido-

[4,5-b]indole-2-carboxamide (19). Following the procedure for the

synthesis of compound 18 from the intermediate 53, compound 19

was obtained with tert-butyl (2-(prop-2-yn-1-yloxy)ethyl)carbamate as

1

the linker instead of t-butyl hex-5-yn-1-ylcarbamate. H NMR (400

MHz, DMSO-d₆) δ (ppm) 12.24 (s, 1H), 11.10 (s, 1H), 9.27 (s, 1H),

P

DOI: 10.1021/acs.jmedchem.6b01816

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

8.23 (t, J = 5.6 Hz, 1H), 7.73−7.68 (m, 3H), 7.57 (s, 1H), 7.35 (s,

1H), 5.91 (s, 1H), 5.11 (dd, J = 12.8 Hz, J = 5.2 Hz, 1H), 3.94 (q, J =

7.2 Hz, 2H), 3.83 (s, 3H), 3.50−3.43 (m, 6H), 3.09 (t, J = 7.6 Hz,

2H), 2.92−2.82 (m, 1H), 2.61−2.46 (m, 2H), 2.30 (s, 3H), 2.10 (s,

3H), 2.05−2.02 (m, 2H), 1.93−1.82 (m, 3H), 1.29 (t, J = 7.2 Hz, 3H),

0.84−0.79 (m, 2H), 0.63−0.59 (m, 2H). UPLC−MS calculated for

C₄₃H₄₅N₁₀O₈[M + H]⁺: 829.34, found 829.41. Purity, >99.0%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(5-(2-(2,6-dioxopiperidin-3-yl)-1,3-di-

oxoisoindolin-4-yl)pentyl)-6-methoxy-9H-pyrimido[4,5-b]-

indole-2-carboxamide (20). Following the procedure used in the

synthesis of compound 18 from the intermediate 53, compound 20

was obtained with tert-butyl pent-4-yn-1-ylcarbamate as the linker

instead of tert-butyl hex-5-yn-1-ylcarbamate. ¹H NMR (400 MHz,

DMSO-d₆) δ (ppm) 12.22 (s, 1H), 11.10 (s, 1H), 9.29 (s, 1H), 8.19

(t, J = 5.6 Hz, 1H), 7.76−7.71 (m, 3H), 7.54 (s, 1H), 7.35 (s, 1H),

5.93 (s, 1H), 5.12 (dd, J = 13.2 Hz, J = 5.2 Hz, 1H), 3.95 (q, J = 7.2

Hz, 2H), 3.82 (s, 3H), 3.29−3.24 (m, 2H), 3.06 (t, J = 8.0 Hz, 2H),

2.92−2.83 (m, 1H), 2.61−2.55 (m, 2H), 2.30 (s, 3H), 2.10 (s, 3H),

2.05−2.00 (m, 1H), 1.90−1.85 (m, 1H), 1.67−1.61 (m, 2H), 1.57−

1.52 (m, 2H), 1.41−1.35 (m, 2H), 1.31 (t, J = 7.2 Hz, 3H), 0.85−0.81

(m, 2H), 0.63−0.59 (m, 2H). UPLC−MS calculated for C₄₃H₄₄N₁₀O₇

[M + H]⁺: 812.34, found 813.33. Purity, 95.3%.

0.78 (m, 2H), 0.62−0.58 (m, 2H). UPLC−MS calculated for

C₄₃H₄₇N₁₀O₇[M + H]⁺: 815.36, found 815.26. Purity, >99.0%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(5-(2-(2,6-dioxopiperidin-3-yl)-1-oxoi-

soindolin-4-yl)pentyl)-6-methoxy-9H-pyrimido[4,5-b]indole-2-

carboxamide (23). See Figures S2 −S4 in Supporting Information.

¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 12.21 (s, 1H), 10.98 (s,

1H), 9.29 (s, 1H), 8.18 (t, J = 5.2 Hz, 1H), 7.57 (d, J = 6.8 Hz, 2H),

7.49−7.43 (m, 2H), 7.35 (s, 1H), 5.93 (s, 1H), 5.13 (dd, J = 13.2 Hz, J

= 5.2 Hz, 1H), 4.49 (d, J = 17.2 Hz, 1H), 4.33 (d, J = 17.2 Hz, 1H),

3.95 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.29−3.24 (m, 2H), 2.96−2.87

(m, 1H), 2.70−2.44 (m, 4H), 2.30 (s, 3H), 2.10 (s, 3H), 2.03−2.00

(m, 1H), 1.88−1.85 (m, 1H), 1.67−1.65 (m, 2H), 1.58−1.54 (m, 2H),

1.39−1.24 (m, 5H), 0.84−0.82 (m, 2H), 0.63−0.59 (m, 2H); ¹³C

NMR (100 MHz, DMSO-d₆) δ (ppm) 172.87, 171.04, 168.37, 165.48,

162.96, 159.17, 156.48, 155.10, 154.07, 152.42, 152.20, 140.49, 137.46,

136.47, 131.88, 131.56, 131.43, 128.26, 120.60, 119.04, 117.43, 113.85,

113.41, 104.51, 98.35, 97.22, 56.01, 51.55, 46.24, 42.41, 31.16, 29.00,

28.80, 26.30, 22.51, 14.86, 11.28, 10.34, 9.58, 7.59. UPLC−MS

calculated for C₄₃H₄₇N₁₀O₆[M + H]⁺: 799.37, found 799.19. Purity,

98.9%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoi-

soindolin-4-yl)butyl)-6-methoxy-9H-pyrimido[4,5-b]indole-2-

General Procedure for Synthesis of Compounds 21−25 and

27. NBS (17.09 g, 96 mmol, 1.2 equiv) and BPO (1.938 g, 8.0 mmol,

0.1 equiv) were added to a stirred solution of methyl 3-bromo-2-

methylbenzoate 57 (18.33 g, 80 mmol, 1.0 equiv) in benzene (150

mL). The solution was heated at reﬂux for 6 h. After cooling to room

temperature, the solvent was evaporated and the residue was puriﬁed

by ﬂash column chromatography with hexane/EtOAc to aﬀord the

intermediate 58 (22.17 g, 90% yield) as a slightly yellow solid.

Compound 38 (14.48 g, 88 mmol, 1.22 equiv) and TEA (13.38 mL,

96 mmol, 1.33 equiv) were added to a stirred solution of compound

58 (22.17 g, 72 mmol, 1.0 equiv) in MeCN (150 mL). The solution

was stirred at 80 °C for 12 h and then cooled to room temperature,

and most of the solvent was evaporated. EtOAc (200 mL) and H₂O

(200 mL) were added to the residue and the solution was ﬁltered to

1

carboxamide (24). H NMR (400 MHz, DMSO-d₆) δ (ppm) 12.22

(s, 1H), 10.96 (s, 1H), 9.29 (s, 1H), 8.23 (t, J = 6.0 Hz, 1H), 7.58−

7.44 (m, 4H), 7.36 (s, 1H), 5.92 (s, 1H), 5.11 (dd, J = 13.2 Hz, J = 5.2

Hz, 1H), 4.47 (d, J = 17.2 Hz, 1H), 4.32 (d, J = 17.2 Hz, 1H), 3.94 (q,

J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.35−3.32 (m, 2H), 2.94−2.84 (m, 1H),

2.69 (t, J = 7.6 Hz, 2H), 2.56−2.35 (m, 2H), 2.30 (s, 3H), 2.10 (s,

3H), 2.01−1.96 (m, 1H), 1.87−1.80 (m, 1H), 1.67−1.54 (m, 4H),

1.29 (t, J = 7.2 Hz, 3H), 0.84−0.79 (m, 2H), 0.62−0.58 (m, 2H).

UPLC−MS calculated for C₄₂H₄₅N₁₀O₆[M + H]⁺: 785.35, found

785.32. Purity, 94.2%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(3-(2-(2,6-dioxopiperidin-3-yl)-1-oxoi-

soindolin-4-yl)propyl)-6-methoxy-9H-pyrimido[4,5-b]indole-2-

1

aﬀord the crude product as a purple solid 59 (17.4 g, 75% yield). H

carboxamide (25). H NMR (400 MHz, MeOD-d₄) δ (ppm) 7.59

NMR (400 MHz, DMSO-d₆) δ (ppm) 11.00 (s, 1H), 7.87 (d, J = 7.6

Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 5.15 (dd, J

= 13.2 Hz, J = 5.2 Hz, 1H), 4.42 (d, J = 17.6 Hz, 1H), 4.27 (d, J = 17.6

Hz, 1H), 2.96−2.87 (m, 1H), 2.62−2.42 (m, 2H), 2.07−2.00 (m, 1H);

¹³C NMR (100 MHz, DMSO-d₆) δ (ppm) 173.30, 171.32, 167.69,

142.58, 135.10, 134.41, 130.98, 122.96, 117.79, 52.22, 48.46, 31.66,

22.74. UPLC−MS calculated for C₁₃H₁₂BrN₂O₃[M + H]⁺: 323.00,

found 322.90.

(d, J = 7.2 Hz, 1H), 7.50−7.43 (m, 3H), 7.25 (s, 1H), 5.98 (s, 1H),

5.09 (dd, J = 13.2 Hz, J = 4.8 Hz, 1H), 4.48 (d, J = 17.2 Hz, 1H), 4.42

(d, J = 17.2 Hz, 1H), 4.13 (q, J = 6.8 Hz, 2H), 3.86 (s, 3H), 3.50−3.40

(m, 2H), 2.83−2.61 (m, 4H), 2.45−2.25 (m, 4H), 2.25−1.91 (m, 7H),

1.43 (t, J = 7.2 Hz, 3H), 0.92−0.89 (m, 2H), 0.68−0.66 (m, 2H).

UPLC−MS calculated for C₄₁H₄₃N₁₀O₆[M + H]⁺: 771.34, found

771.35. Purity, >99.0%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-6-methoxy-N-(5-(2-(1-methyl-2,6-dioxo-

piperidin-3-yl)-1-oxoisoindolin-4-yl)pentyl)-9H-pyrimido[4,5-

b]indole-2-carboxamide (26). K₂CO₃(332 mg, 2.4 mmol, 1.2

equiv) and MeI (0.19 mL, 3.0 mmol, 1.5 equiv) were added to a

solution of compound 59 (646 mg, 2.0 mmol, 1.0 equiv) in DMF (5.0

mL). The solution was stirred at 60 °C for 3 h. Preparative HPLC

puriﬁcation aﬀorded the desired compound 63 as a white solid (485

mg, 72% yield). ¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 7.86 (d, J =

8.4 Hz, 1H), 7.78 (d, J = 7.2 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 5.22

(dd, J = 13.2 Hz, J = 5.2 Hz, 1H), 4.41 (d, J = 17.6 Hz, 1H), 4.26 (d, J

= 17.6 Hz, 1H), 3.06−2.95 (m, 4H), 2.79−2.73 (m, 1H), 2.52−2.41

(m, 1H), 2.07−2.01 (m, 1H); ¹³C NMR (100 MHz, DMSO-d₆) δ

(ppm) 172.33, 170.94, 167.73, 142.60, 135.11, 134.38, 130.96, 122.99,

117.79, 52.69, 48.41, 31.80, 27.06, 22.05. UPLC−MS calculated for

C₁₄H₁₄BrN₂O₃[M + H]⁺: 337.02, found 336.98.

Following the procedures used to prepare compound 18 from the

intermediate 53, compounds 21−25 and 27 with diﬀerent alkynyl

chains were obtained as indicated in Schemes 8 and 9.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(6-(2-(2,6-dioxopiperidin-3-yl)-1-oxoi-

soindolin-4-yl)hexyl)-6-methoxy-9H-pyrimido[4,5-b]indole-2-

1

carboxamide (21). H NMR (400 MHz, DMSO-d₆) δ (ppm) 12.25

(s, 1H), 10.97 (s, 1H), 9.31 (s, 1H), 8.16 (t, J = 5.2 Hz, 1H), 7.60−

7.21 (m, 5H), 5.95 (s, 1H), 5.13 (dd, J = 13.2 Hz, J = 4.8 Hz, 1H),

4.48 (d, J = 17.2 Hz, 1H), 4.32 (d, J = 17.2 Hz, 1H), 3.95 (q, J = 7.2

Hz, 2H), 3.83 (s, 3H), 3.29−3.24 (m, 2H), 2.94−2.87 (m, 1H), 2.73−

2.45 (m, 4H), 2.30 (s, 3H), 2.10 (s, 3H), 2.03−1.98 (m, 1H), 1.89−

1.82 (m, 1H), 1.66−1.62 (m, 2H), 1.52−1.48 (m, 2H), 1.36−1.27 (m,

7H), 0.84−0.79 (m, 2H), 0.63−0.59 (m, 2H). UPLC−MS calculated

for C₄₄H₄₉N₁₀O₆[M + H]⁺: 813.38, found 813.29. Purity, 95.3%.

4-((3-Cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-di-

methylisoxazol-4-yl)-N-(2-(3-(2-(2,6-dioxopiperidin-3-yl)-1-ox-

oisoindolin-4-yl)propoxy)ethyl)-6-methoxy-9H-pyrimido[4,5-

Following the procedures used for the conversion of the

intermediates 53−18, compound 26 was obtained from the

1

intermediate 63. H NMR (400 MHz, MeOD-d₄) δ (ppm) 7.56−

b]indole-2-carboxamide (22). H NMR (400 MHz, DMSO-d₆) δ

7.54 (m, 2H), 7.43−7.36 (m, 3H), 6.20 (s, 1H), 5.12 (dd, J = 13.6 Hz,

J = 5.2 Hz, 1H), 4.47 (d, J = 17.2 Hz, 1H), 4.41 (d, J = 17.2 Hz, 1H),

4.22 (q, J = 6.8 Hz, 2H), 3.89 (s, 3H), 3.50−3.40 (m, 2H), 3.07 (s,

3H), 2.90−2.81 (m, 2H), 2.72−2.61 (m, 2H), 2.51−2.40 (m, 1H),

2.32 (s, 3H), 2.16 (s, 3H), 2.13−2.09 (m, 1H), 2.03−1.99 (m, 1H),

1.70−1.66 (m, 4H), 1.48−1.44 (m, 5H), 1.10−1.05 (m, 2H), 0.83−

(ppm) 12.23 (s, 1H), 10.96 (s, 1H), 9.27 (s, 1H), 8.23 (t, J = 5.2 Hz,

1H), 7.57−7.41 (m, 4H), 7.35 (s, 1H), 5.90 (s, 1H), 5.10 (dd, J = 13.2

Hz, J = 5.2 Hz, 1H), 4.47 (d, J = 17.2 Hz, 1H), 4.32 (d, J = 17.2 Hz,

1H), 3.93 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.50−3.40 (m, 6H), 2.88−

2.84 (m, 1H), 2.71 (t, J = 7.6 Hz, 2H), 2.67−2.43 (m, 2H), 2.30 (s,

3H), 2.10 (s, 3H), 1.98−1.81 (m, 4H), 1.29 (t, J = 7.2 Hz, 3H), 0.83−

Q

DOI: 10.1021/acs.jmedchem.6b01816

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

0.81 (m, 2H). UPLC−MS calculated for C₄₄H₄₉N₁₀O₆[M + H]⁺:

813.38, found 813.34. Purity, >99.0%.

Flow cytometry was used to analyze eﬀects of the drugs on cell cycle

(propidium iodide staining) and apoptosis (annexin V and propidium

iodide staining). Cell were treated with compounds at the indicated

concentrations for 24 h, collected, stained, and analyzed by ﬂow

cytometry.

7-(3,5-Dimethylisoxazol-4-yl)-N-(5-(2-(2,6-dioxopiperidin-3-

yl)-1-oxoisoindolin-4-yl)pentyl)-6-methoxy-9H-pyrimido[4,5-

1

b]indole-2-carboxamide (27). H NMR (400 MHz, DMSO-d₆) δ

For Western blot analysis, 2 × 10⁶cells/well were treated with

compounds at the indicated concentrations for various times. Cells

were collected and lysed in RIPA buﬀer containing protease inhibitors.

An amount of 20 μg of lysate was run in each lane of a PAGE−SDS

and blotted into PVDF membranes. Antibodies for immunoblotting

were BRD2, BRD3, and BRD4 purchased from Bethyl Laboratories

(Montgomery, TX, USA), c-Myc from Cell Signaling Technology

(Danvers, MA, USA), and GAPDH from Santa Cruz Biotechnologies

(Dallas, TX, USA).

Eﬃcacy and Pharmacodynamics Studies in the RS4;11

Xenograft Model in Mice. All animal experiments were done

under the guidelines of the University of Michigan Committee for Use

and Care of Animals and using an approved animal protocol

(PRO00005315, PI, Shaomeng Wang).

(ppm) 12.57 (s, 1H), 10.97 (s, 1H), 9.60 (s, 1H), 9.01 (t, J = 6.0 Hz,

1H), 8.12 (s, 1H), 7.56−7.54 (m, 2H), 7.52 (s, 1H), 7.48−7.41 (m,

2H), 5.13 (dd, J = 13.2 Hz, J = 5.2 Hz, 1H), 4.48 (d, J = 17.2 Hz, 1H),

4.32 (d, J = 17.2 Hz, 1H), 3.91 (s, 3H), 3.38−3.33 (m, 2H), 2.96−2.87

(m, 1H), 2.68−2.57 (m, 3H), 2.51−2.42 (m, 1H), 2.32 (s, 3H), 2.12

(s, 3H), 2.07−1.99 (m, 1H), 1.69−1.60 (m, 4H), 1.44−1.36 (m, 2H).

UPLC−MS calculated for C₃₅H₃₆N₇O₆[M + H]⁺: 650.27, found

650.26. Purity, >99.0%.

7-(3,5-Dimethylisoxazol-4-yl)-6-methoxy-N-methyl-9H-

pyrimido[4,5-b]indole-2-carboxamide (28). 10% Pd/C (20 mg)

was added to a stirred solution of compound 33 (200 mg, 0.52 mmol,

1.0 equiv) in 10 mL of DMF under N₂. The ﬂask was purged and

reﬁlled with H₂three times. The solution was then stirred at 60 °C for

12 h. After cooling to room temperature, the solution was ﬁltered

through Celite. After concentration, the residue crude intermediate 35

was suspended in THF (30 mL), and LiOH (38 mg, 1.55 mmol, 3.0

equiv) was added. The resulting solution was stirred at room

temperature for 12 h. After concentration, the residue was puriﬁed

by preparative HPLC to aﬀord the compound 36 as a yellow solid (52

mg, 30% yield for two steps). UPLC−MS calculated for C₁₇H₁₅N₄O₄

[M + H]⁺: 339.11, found 339.08.

Compound 36 (150 mg, 0.44 mmol) was suspended in thionyl

chloride (0.64 mL, 8.8 mmol), and two drops of dry DMF were added.

The mixture was stirred at reﬂux for 2 h. Excess thionyl chloride was

removed in vacuo, and the resulting solid was dried. The resulting acyl

chloride was used without further puriﬁcation. Dry THF (15 mL) was

added to the acyl chloride. The mixture was cooled to 0 °C, and

subsequently, methylamine hydrochloride (45 mg, 0.67 mmol) was

added. After stirring at room temperature for 2 h, the mixture was

evaporated and puriﬁed by preparative HPLC to get 61.8 mg of

compound 28 with a yield of 40%. ESI-MS calculated for C₁₈H₁₇N₅O₃

[M + H]⁺= 52.13. Obtained: 352.17. ¹H NMR (400 MHz, MeOD-d₄)

δ 9.55 (s, 1H), 8.03 (s, 1H), 7.54 (s, 1H), 3.96 (s, 3H), 2.35 (s, 3H),

2.18 (s, 3H), 1.31 (t, J = 7.3 Hz, 3H). UPLC analysis (10 min from

10% to 100% (MeCN/H₂O containing 0.1% CF₃CO₂H)): retention

time, 2.70 min; peak area, 98.12%.

To develop xenograft tumors, 5 × 10⁶RS4;11 cells with 50%

Matrigel were injected subcutaneously on the dorsal side of severe

combined immunodeﬁcient (SCID) mice, obtained from Charles

River, one tumor per mouse. When tumors reached ∼100 mm³, mice

were randomly assigned to treatment and vehicle control groups.

Animals were monitored daily for any signs of toxicity and weighed 2−

3 times per week during the treatment and weighed at least weekly

after the treatment ended. Tumor size was measured 2−3 times per

week by electronic calipers during the treatment period and at least

weekly after the treatment was ended. Tumor volume was calculated as

V = LW²/2, where L is the length and W is the width of the tumor.

For pharmacodynamic analysis, resected RS4;11 xenograft tumor

tissues were ground into powder in liquid nitrogen and lysed in lysis

buﬀer (1% CHAPS, 150 mM NaCl, 20 mM Tris-HCl, 1 mM. EDTA, 1

mM EGTA, and COMPLETE proteinase inhibitor (Roche)). Whole

tumor lysates were separated on 4−20% Novex gels. The separated

proteins were transferred to a polyvinylidene diﬂuoride membrane for

immunoblotting. The following antibodies were used: rabbit

polyclonal antibodies for BRD2 (A302-583A), BRD3 (A302-368A),

and BRD4 (A301-985A100) from Bethyl Laboratories; c-Myc

(D84C12), PARP (46D11), and caspase-3 (8G10) from Cell Signaling

Technology, and actin goat polyclonal antibody from Santa Cruz

Biotechnology. The secondary antibody used was horseradish

peroxidase conjugated goat anti-rabbit (Thermo Scientiﬁc). The

BIO-RAD Clarity Western Enhanced Chemiluminescence Substrates

and HyBlot Chemiluminescence ﬁlm were used for signal develop-

ment and detection using a SRX-101A tabletop processor (Konica

Minolta).

Determination of Biochemical Binding Aﬃnities to BET

Proteins. Binding aﬃnities of BET inhibitors to BRD2 (BD1 and

BD2 proteins), BRD3 (BD1 and BD2 proteins), BRD4 (BD1 and

BD2 proteins) were determined using our established ﬂuorescence-

polarization binding assays as described previously.²⁷

Cell Growth Inhibition, Apoptosis Analysis, and Western

Blotting. The human acute leukemia RS4;11 cell line (CRL-1873)

was purchased from the American Type Culture Collection, and the

human acute leukemia MOLM-13 cell line was purchased from the

DSMZ German cell bank (ACC554). In all experiments, RS4;11 and

MOLM-13 human leukemia cells were used within three months of

thawing fresh vials. RS4;11 and MOLM-13 cells were cultured in

RPMI 1640 media supplemented with 10% FBS and 1% penicillin−

streptomycin at 37 °C in a humidiﬁed atmosphere containing 5% CO₂

in air.

In cell growth experiments, cells were seeded in 96-well cell culture

plates at a density of 10000−20000 cells/well in 100 μL of culture

medium. Each compound tested was serially diluted in the appropriate

medium, and 100 μL of the diluted solution containing the tested

compound was added to the appropriate wells of the cell plate. After

addition of the tested compound, the cells were incubated for 4 days at

37 °C in an atmosphere of 5% CO₂. Cell growth was evaluated by a

lactate dehydrogenase-based WST-8 assay (Dojindo Molecular

Technologies) using a Tecan Inﬁnite M1000 multimode microplate

reader (Tecan, Morrisville, NC). The WST-8 reagent was added to the

plate, incubated for at least 1 h, and read at 450 nm. The readings were

normalized to the DMSO-treated cells, and the IC₅₀was calculated by

nonlinear regression analysis using GraphPad Prism 6 software.

Determination of Drug Concentrations in Plasma and

RS4;11 Tumor Tissue. Pharmacokinetics of compound 23 was

determined in female SCID C.B-17 mice bearing RS4;11 tumors

following a single intravenous dose of 5 mg/kg. Compound 23 was

dissolved in the vehicle containing 20% (v/v) polyethylene glycol 400,

6% (v/v) Cremophor EL and 74% (v/v) PBS (20% PCP). Mice were

sacriﬁced at 1, 3, 6, and 24 h after drug treatment and at 6 h after

vehicle treatment, followed by collection of blood samples (300 μL)

and tumor samples. Blood samples were centrifuged at 15 000 rpm for

10 min, then the supernatant plasma was saved for analysis. Isolated

tumor samples were immediately frozen and ground with a mortar and

pestle in liquid nitrogen. All plasma and tumor samples were stored at

−80 °C prior to analysis.

Plasma and tumor concentrations of compound 23 were

determined by a LC−MS/MS method developed and validated for

this study. The LC−MS/MS method, consisting of a Shimadzu HPLC

system and chromatographic separation of tested compound, was

achieved using a Waters XBridge-C18 column (5 cm × 2.1 mm, 3.5

μm). An AB Sciex QTrap 5500 mass spectrometer equipped with an

electrospray ionization source (Applied Biosystems, Toronto, Canada)

in the positive-ion multiple reaction monitoring (MRM) mode was

used for detection. The mobile phases were 0.1% formic acid in

puriﬁed water (A) and 0.1% formic acid in acetonitrile (B). The

R

DOI: 10.1021/acs.jmedchem.6b01816

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

gradient (B) was held at 10% (0−0.3 min), increased to 95% at 0.7

min, then held at isocratic 95% B for 2.3 min and then immediately

stepped back down to 10% for 2 min re-equilibration. The ﬂow rate

was set at 0.4 mL/min.

Cancer Institute, NIH (Grant P30CA046592), and Medsyn

Biopharma.

ABBREVIATIONS USED

■

In Vitro Metabolite Identiﬁcation in Mouse Liver Micro-

somes. Compound 23 (10 μM) was incubated with mouse liver

microsomes (MLM) and β-NADPH at 37 °C for 20 and 40 min. The

reaction was quenched by adding a 3-fold volume of ice-cold

acetonitrile. The mixture was centrifuged at 15 000 rpm for 10 min,

and the supernatant was saved under −80 °C for analysis. The

negative control samples were prepared by a similar procedure without

NADPH or using boiled microsomes. The general approach for

metabolite identiﬁcation using AB Sciex QTrap 5500 mass

spectrometer involves the following steps: (1) Obtain a product ion

spectrum of the parent compound to establish fragmentation. (2)

Interpret the spectrum to identify major fragment ion and possible

neutral loss. (3) Collect spectra of samples using both established

precursor ion scan and neutral loss scan. EMS scan of both control and

samples were acquired also. (4) Run product ion scans and MRM

scans for all possible metabolite identiﬁed from step 3 plus expected

metabolite. (5) Interpret the spectrum of the metabolites and

determine the structure with their logical fragmentation pattern.

AML, acute myeloid leukemia; BD1, bromodomain 1; BD2,

bromodomain 2; BET, bromodomain and extra-terminal

domain; BINAP, 2,2′-bis(diphenylphosphino)-1,1′-binaphtha-

lene; BRD2, bromodomain-containing protein 2; BRD3,

bromodomain-containing protein 3; BRD4, bromodomain-

containing protein 4; BRDT, bromodomain testis-speciﬁc

protein; FP, ﬂuorescence polarization; GAPDH, glyceraldehyde

3-phosphate dehydrogenase; MLL1, mixed lineage leukemia

protein 1; MRM, multiple reaction monitoring; NUT, nuclear

protein in testis; PBS, phosphate buﬀered saline; PD,

pharmacodynamics; PK, pharmacokinetics; PROTAC, proteol-

ysis targeting chimera

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ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.6b01816.

■

S

Western Blotting analysis of BRD2, BRD3, and BRD4

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AUTHOR INFORMATION

Corresponding Author

*Phone: 1-734-615-0362. Fax: 1-734-647-9647. E-mail:

shaomeng@umich.edu.

■

ORCID

Bing Zhou: 0000-0003-1813-8035

Shaomeng Wang: 0000-0002-8782-6950

(9) Berthon, C.; Raffoux, E.; Thomas, X.; Vey, N.; Gomez-Roca, C.;

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Author Contributions

^∇B.Z., J.H., F.X., Z.C., L.B., E.F.-S., and M.L. contributed

equally.

Notes

The authors declare the following competing ﬁnancial

interest(s): S. Wang, B. Zhou, F. Xu, J. Hu, L. Bai, C.-Y.

Yang, D. McEachern, and S. Przybranowski are co-inventors for

BET degraders disclosed in this study. The University of

Michigan has ﬁled a number of patent applications related to

these BET degraders, which have been licensed to Medsyn

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ACKNOWLEDGMENTS

■

This study is supported in part by funding from the Breast

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Michigan Comprehensive Cancer Center Strategic Fund for

Breast Cancer (to S.W.), the University of Michigan

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DOI: 10.1021/acs.jmedchem.6b01816

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of a Small-Molecule Degrader of Bromodomain and Extra-Terminal (BET) Proteins with Picomolar Cellular Potencies and Capable of Achieving Tumor Regression

DOI: 10.1021/acs.jmedchem.6b01816

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